OXIDATION AND EVOLUTION OFMOLECULAR HYDROGEN …aspect of intermediary metabolism with our leased in...

OXIDATION AND EVOLUTION OF MOLECULAR HYDROGEN BYMICROORGANISMS

HOWARD GESTDepartment of Microbiology, Western Reserve University, School of Medicine, Cleveland, Ohio

CONTENTSI. Autotrophic organism .................................................................... 44

1. Photosynthetic microorganisms .......................................................... 442. The hydrogen bacteria.................................................................. 453. Sulfate reducing bacteria................................................................ 46

II. Heterotrophic organism ................................................................... 461. The clostridia ......................................................................... 46

A. Carbohydrates and related compounds as precursors and acceptors of H. .............. 46a. The importance of metals......................................................... 47b. Clostridium kluyvers ............................................................... 47c. Clostridium aceticum............................................................... 48

B. Organic nitrogen compounds as precursors and acceptors of H ........................ 482. Veillonella gazogenes (Micrococcue lactilyticus); M. aerogenes.............................. 493. Diplococcus glycinophilus ................................................................ 504. The methane bacteria................................................................... 505. The colon-aerogenes bacteria............................................................ 52

A. Influence of nutritional factors on H, metabolism.................................... 53a. Oxygen........................................................................... 53b. Formate and fermentable carbohydrates ........................................... 53c. Amino acids and ammonia......................................................... 54d. Nitrate and fumarate.............................................................. 55e. Metals............................................................................ 56f. Other factors..................................................................... 57

B. Mechanism of the hydrogenylase reaction............................................ 57C. The exchange reaction............................................................... 61D. Oxidation of H2 with various acceptors.............................................. 62

a. Fumarate ......................................................................... 62b. Nitrate........................................................................... 63c. Methylene blue and other acceptors............................................... 64

6. Hydrogenase in organisms which do not oxidize nor produce H2 during "normal" metab-olism.................................................................................... 65

The pathways through which electrons are tracer techniques. At the same time, there havetransported in the respiratory and fermentative been no comparable new developments whichprocesses of microorganisms are to a large degree could form the basis for a more simplified andstill uncharted. A steadily increasing number of refined approach to the study of electron path-individual reactions are being examined from this ways. This state of affairs is not unique to mi-standpoint, but there is no doubt that we are crobial metabolism but could equally well be saidand will be for sometime in the "qualitative" of the metabolism of more complex organisms.stage of exploration. This is particularly evident Among microorganisms, particularly in thewhen we compare present knowledge of this vital facultative and strict anaerobes, the electrons re-aspect of intermediary metabolism with our leased in oxidative reactions can be transferredrapidly growing detailed understanding of the to a variety of ultimate acceptors, and frequentlyfate of carbon, nitrogen, and phosphorus in several alternative routes are available in anumerous complicated reaction sequences. single organism. In addition to the potentiality ofRapid progress in the latter area has been facili- using diverse electron acceptors, many micro-tated by the availability and application of sensi- organims can perform oxidations in the absencetive methods such as the chromatographic and of "external" acceptors by the simple expedient

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44 HOWARD GEST [voL 18

of producing molecular hydrogen. Elementary ous other species of green, blue-green, red, andhydrogen also can be activated by numerous organ- brown algae can utilize H2 for photochemicalisms and as a consequence can serve as a re- reduction of CO2.ductant in certain types of biochemical con- Light dependent formation of H2 by algae isversions. One of the catalysts presumed to be observed in the absence of added organic com-common to the complex systems responsible for pounds (3) which suggests that water may be theevolution and oxidation of H2 is the enzyme ultimate electron source. This interpretation is"hydrogenase". The ability to catalyze an ex- supported by the reported inability of organicchange reaction between water and D2 is also compounds to augment the rate of the reaction.attributed to this enzyme and is in fact believed It is by no means excluded, however, that theto be one of its fundamental properties. Through actual H2 precursor(s) may be organic-in thesecommon usage the term "hydrogenase activity" instances, compounds which are derived fromhas become synonymous with either exchange or endogenous reserve materials (4).reducing activity. The "photoheterotrophs" which produce H2A survey of the occurrence of H2 metabolism are notably the sulfur purple and nonsulfur

in microorganisms discloses that the ability to purple bacteria. Resting cells of these organisms,evolve or utilize H2 (or to catalyze the exchange in certain instances, evolve H, in dark fermenta-reaction) is not a restricted metabolic potentiality tions of glucose, Ca compounds, and formatebut rather that this type of activity is found in (5, 6). In addition, a rapid photochemical pro-a large number of organisms of widely different duction of Ho occurs in the presence of suitablephysiological types. This fact alone suggests that organic substrates if the gas atmosphere aboveH2 metabolism represents a fundamental aspect the organisms does not contain N2 (7, 8). Theof microbial physiology. In this review an at- inhibition of photoevolution of H, by N, (andtempt has been made to summarize, correlate, NH') was the major clue to the discovery thatand evaluate the surprisingly extensive literature photosynthetic bacteria possess "nitrogenase"dealing with the metabolism of Hs in micro- which enables them to use N2 as the sole sourceorganisms. Special emphasis has been accorded of nitrogen for growth. A suggested, but as yetthe colon-aerogenes bacteria since these organism unproved, mechanism of the inhibition is coin-represent the most thoroughly studied group petition for metabolic hydrogen between hy-in this regard. drogenase and reductive reactions in the conver-

sion of N2 to the amino group (7).PART I. AIJTOTROPHIO ORGANISMS Molecular hydrogen is formed as a major

1. PHOTOSYNTHETIC MICROORGANISMS metabolic product by photosynthetic bacteria

A variety of autotrophic organismsevolve or during growth under certain nutritional cir-

utilize H2. Evolution of H, in this group appears instances. One of the most important factorsto be mainly restricted to the photosynthetic in this respect is the type of the nitrogen source;autotrophs and the s-called "photohetero- hydrogen is not produced when the source is antrophs". In the former category are green alga am u salt orpwhen high concentrations ofsuch as Scenedemus, which slowly produces H, yeas extract, peptone o rhsimilar complexin the dark and at a relatively rapid rate upon materals are present. Molecular hydrogen is aillumination (1). In Sceeemus, H, metabolismm characteristic product, however, when aminoappemiaronly aftr an extended dar metaneobic acids such as glutamic and aspartic constitute theappears only after an extended dark anaerobic niroe supyA Cranaio cd evadaptation period. Once the orgamni is nitrogensupply (9). Certai amio acids serveit is not only capable of photochemical H, pro- as excellent nitrogen sources but do not permitduction but can also reduce C02 with H, either He evolution during growth, presumably becausephotosynthetically or by a dark chemoauto- hey give to a high stedy state concentrationtrophic process, in which the energy for C02 of ammoni i the medium (10).assimilation is derived from oxidation of H2 The direct precursor of H, in the light de-with 02. The latter type of oxidation is the energy pendent process in bacteria is unknown. It seemssource for growth of the autotrophic hydrogen quite possible that here again water may not bebacteria which are discussed below. Frenkel the immediate source, but rather that H2 isand Rieger (2) have recently shown that numer- evolved from organic intermediates (e.g., formate


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1954] OXIDATION AND EVOLUTION OF MOLECULAR HYDROGEN 45

or pyruvate) by m anisms simila to those Thus, they can grow either heterotrophically onoperating in gas producing heterotrophs [see a variety of organic compounds or autotrophicallydiscussion in (7)]. The light dependency may, m a with C02 as the sole source of carbon, growthcertain sense, be secondary, i.e., light could be energy with the latter usually being supplied byrequired for the generation of an organic pre- the "Knailgas reaction":cursor which is subsequently oxidized in a dark 2H2 + 02 =2H20reaction, the electrons being transferred to thehydrogenase system. Previous work with these bacteria was mainly

Resting cells of photosynthetic bacteria can concerned with determination of the stoichi-oxidize Hs in the dark with a variety of ac- ometry of the gas exchanges and calculation ofceptors, e.g., oxygen, nitrate, sulfate, thiosulfate, the thermodynamic efficiency of the over-allferricyanide, fumarate, dyes (5, 11, 12, 13). With chemosynthetic process. As a consequence ofC02, however, H, is not utilized unless light of these emphases little is known of the intermediarythe proper wavelengths is supplied. These ob- metabolism of these interesting organis Oneservations suggest that the activity of hydro- outstanding characteristic is the adaptive naturegenase, per se, in these organisms is not light of the hydrogenase system; when cultivated independent, a conclusion supported by the finding organic media as heterotrophs, most speciesthat the activity of the cell-free hydrogenase of generally lose or at least have a diminishedRhodopirillum rubrum is unaffected by illumina- capacity for performing the Knaligas re-tion (13).1 When CO, is the ultimate oxidant, action (15).light may be obligatory for the generation of One species, Hydrogenomonas aqia, is reportedoxidized acceptors which are reduced by H, in to be capable of growing autotrophically withdark reactions (i.e., the converse of the argument nitrate as the electron acceptor in place of oxygenused in explaining the light dependence of H, (16). Nitrate is also reduced (to nitrite) as aevolution). result of hydrogenase action by resting cells of a

It is likely that photoproduction of H, has an newly described species, Hydrogenomona facilisimportant significance for the mechanism of (17). Although this organism reduces oxygenelectron transport in all types of photosynthetic and methylene blue actively, other possible ac-reactions. From the very existence of light ceptors including acetaldehyde, fumarate,dependent H, evolution, it may be inferred that pyruvate, oxalacetate, and a-ketoglutarate dothe photochemical generation of electrons (re- not serve as oxidants for Hs. Molecular hydrogenducing power) occurs in a reaction characterized and lactate are simultaneously oxidized by rest-by a redox potential well below those of the ing s ons of Hydrogenomonas (15, 18)pyridine nucleotide coenzyme systems and, ac- which suggests that the terminal electron trans-cordingly, that these coensymes are probably port enzymes are usually not rate-limiting.not reduced by "primary" acts. In this connec- It should be noted that hydrogenase activitytion, it is tempting to speculate that the carriers with oxygen and nitrate as acceptors occurs ininvolved in the early stages of electron transport various heterotrophic organisms, but in these thein photosynthesis may be similar to those par- oxidation of Hs presumably does not constitute aticipating in the phosphoroclastic reaction of the major energy yielding reaction with respect toclostridia and in decomposition of formate by the growth. Nevertheless, it seems likely that thehydrogenlyase complex (see sections II. 1,A and basic mechanism of H, activation and oxidationII. 5,B). is similar, if not identical, in autotrophs and

heterotrophs. Of considerable interest is the2. THE HYDROGEN BACTERIA report that conversion of inorganic phosphate

These organisms are generally facultative with to energy rich phosphate compounds occursregard to the autotrophic mode of development. during catalysis of the Knailgas reaction by

crude cell-free extracts of the heterotroph,The properties of the cell-free hydrogenase Arutobcelreenexacts(9 thi sytemoappafrom Rhodospirillum rubrum appear to be similarto those of the corresponding enzyme from Each- potentially useful both as a model for energyerichia coli (13, 14). The latter enzyme is discussed metabolism in chemosynthesis and for studiesin section II. 5, D. on the mechanism of oxidative phosphorylation.


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46 HOWARD GEST [vo. 18

3. SULFATEtREDUCING BACTERIA From several points of view it is convenient

Until recently, it was believed that the micro- to consider the level of hydrogenate activity asorganisms responsible for reduction of sulfate the factor which controls the disposition ofto sulfide in soil and other natural environments electrons into possible alternative routes. Inwere obligate heterotrophs. The work of Starkey this sense, hydrogenase could be considered asand Wight (20) made it probable, however, that a substitute for cytochrome enzymes, whichautotrophic sulfate reducers exist which utilize have thus far not been detected in strictH2 rather than organic compounds as the hy- anaerobes. Insufficient data are available, how-drogen donor for this process. They experienced ever, to permit a decision between this viewdifficulty in isolating the orgasms from success- and the possibility that hydrogenase is not theful enrichment cultures and were thus able to actual controlling factor but rather a'leak" whichestablish this "physiological group" only tenta- enables the organism to discard exce electronstively. The (anaerobic) autotrophic growth of A. Carbhydrt and Related Compounds ascertain strains of Desufovibrio desufurican, Precursors and Acceptors of H2originally isolated as heterotrophs, was subse-quently demonstrated by Butlin and co-workers An important H2-evolving process which oc-(21, 22). From a comparative biochemical stand- curs in saccharolytic clostridia is the so-calledpoint, such organisms may be considered as "phosphoroclastic" reaction:"hydrogen bacteria" which have developed theability to use sulfate as the ultimate electron CHCOOPOxH2 + C02 + H2acceptor during growth. The reduction of sulfateto sulfide with H2 as the donor has special sig- The term "phosphoroclastic" as applied to thisnificance for the problem of microbiological reaction is actually a misnomer since this desig-corrosion of iron as described in detail by Starkey nation was coined to describe the cleavage ofand Wight (20; see also 23). pyruvate to acetyl phosphate and formate by

Resting cell suspensions of D. desulfuricans colon-aerogenes bacteria. Although C02 plus H2oxidize H2 with sulfate and also with more re- superficially appear to be the equivalent ofduced inorganic sulfur compounds which are formate, it is possible that these degradations ofsuspected of being intermediates in the produc- pyruvate involve quite different mechanismstion of sulfide (24). It is anticipated that the use (see section II. 5,B). Formate is usually notof organisms with hydrogenase that can link metabolized by intact or cell-free preparationswith the various enzymes involved in sulfate of the clostridia, and it has been shown that thisreduction will in some respects simplify study of compound is not an intermediate in pyruvatethe details of the reaction sequence. breakdown by Clostridium butylicum (26, 27).

Several papers, however, report formate produc-PART II. HETEROTROPHIC ORGANISMS tion from carbohydrates and other substrates by

the clostridia under certain conditions.1. THIE CLOSTRIDIA Reversibility of the "phosphoroclastic" de-

The strict anaerobes do not utilize oxygen as composition is indicated by incorporation ofthe ultimate oxidant during normal metabolism, isotopic C02 into the carboxyl group of pyruvateand the terminal electron acceptor is, conse- (27) and by the inhibiting effect of high H2quently, either another molecule of the substrate pressure on the rate of the forward reactionbeing metabolized, a derivative of the substrate, (28, 29). On the other hand, attempts to demon-or the hydrogenate system. In certain instances, strate an unequivocal incorporation of acetateseveral or all of these acceptor "systems" are or acetylphosphate into pyruvate have not beenused simultaneously, and the quantitative levels successful (27). Cell-free extracts of C. bintylicumof the fermentative end products then reflect which actively catalyze the "phosphoroclastic"the balance among the alternative electron path- reaction can be readily prepared and have beenways (e.g., see 25). This is true, of course, not useful in clarifying the over-all features of theonly of the strict anaerobes such as the clostridia, process (26, 27). Presumably, hydrogenate is anbut also of the facultative organisms when they essential component of the enzyme complex, butmetabolize anaerobically. little study has been made of this aspect of the


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problem. Although Koepsell and Johnson (26) tions contain the enzymes necessary for synthesisnoted that extracts of C. butylicum do not display of higher fatty acids from C2 precursors and alsohydrogenase activity with methylene blue as the display hydrogenase activity with several ac-acceptor, intact cells of C. acetobutylicum do ceptors. The latter activities are represented byexhibit very high hydrogenase activity with this the following equations:dye (30). One possible explanation of this differ-ence is that cell-free preparations are deficient CH.CHO + H2 CHOH,0H (3)in a carrier required for hydrogen transfer to CHCOCH2CO0- +12methylene blue, a view supported by the oh- CHCHOHCH2COO- (39)servation that extracts of C. butylicum oxidize CHCOOPO$- + CH3COO- + 2H2H2 rapidly in the presence of benzyl viologen _ CHCH2CH2COO- + HPO- + H20 (38)(Peck, H. D., and Gest, H., unpublished data).A recent report by Wolfe and OXane (31) sug- CH2CHCH2COO- +112gests that hydrogenase or a closely associated CHH2CH2 (40)enzyme is the most labile component of the cell- RCOOPOs- + 2H2 -+ RCH20H + HPO- (41)free "phosphoroclastic" enzyme system.

a. The importance of mweto. The production where R = 0317, CM., or 0.1113of H12 from glucose by C. butyricum is inhibited From the foregoing, it is evident that H2 canby CO, and the inhibition can be reversed by be oxidized by the C. kluyveri extract with thelight. Kempner and Kubowitz (32) obtained an simultaneous reduction of aldehyde, keto, oraction spectrum of the "H2evolving enzyme" in carboxyl phosphate groups or of a double bond.intact cells by determining the efficiencies of As shown by the fourth equation, vinylacetate isinhibition reversal with light of different wave- reduced by H2 to butyric acid. Recent experi-lengths. Reversibility of the inhibition by light ments of Peel and Barker (42) indicate that theimplicates an iron enzyme, but the action spec- actual substrate reduced in this reaction is thetrum observed is not typical of an iron porphyrin acyl derivative of an unidentified cofactor (pos-(33). In the presence of CO, the fermentation of sibly coenzyme A). Vinylacetate can also beglucose results mainly in lactic acid as opposed oxidized anaerobically by the cell-free prepara-to formation of H2, C02, and volatile acids by tion with the formation of H2 according to theuninhibited cells. A similar shift in the glucose equation:fermentation pattern is observed when other CH2=CHCH2COO0 + HPO + H20metal complexing agents such as cyanide ar e CHsCOOPOs- + CH3COO- + H2added to suspensions of C. telchii (34). The sameresult can also be achieved by controlling the Since the extracts do not produce H2 from addedmetal nutrition of this organism; thus, cells from ethanol or acetaldehyde, Stadtman and Barkera medium rich in iron ferment glucose to H2, C02, (40) suggest that the H1 formed during growthand volatile acids while those from a medium on C2 substrates is derived from vinylacetatedeficient in iron carry out a lactic acid fermenta- or a closely related C4 compound which occurstion (34, 35). All these observations emphasize as an intermediate in butyrate synthesis. It stillthe importance of iron in the formation of H2 seems possible, however, that the precursor ofby clostridia, but the site of action of the metal H2 is a C2 compound such as an acetaldehydeand its actual function are still not clear. Signifi- derivative. Anaerobic oxidation of acetaldehydecantly, a metal, probably iron, is also essential to acetic acid and H2 has been frequently as-in H1 production from formate by colon-aerogenes sumed to be the source of molecular hydrogenbacteria (see section II. 5,Ae). in early fermentation schemes (e.g., see 43):

b. Cloetridium kluyveri. C. kluyveri differs fromtypical saccharolytic clostridia in that it does notferment common sugars or C3 compounds such The free energy of this reaction is large enoughas lactate and pyruvate (36, 37). During growth to account for evolution of molecular hydrogenin a medium containing ethyl alcohol and ace- (44). Production of 12 from pyruvate by or-tate, the organism produces butyric and caproic ganisms of the C. butylicum type may involve aacids and small amounts ofH2. Cell-free prepara- imilar oxidation; decarboxylation of pyruvate


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48 HOWARD GEST [VOL. 18

would give rise to an "acetaldehyde-coenzyme" fermentations of single amino acids (50, 51, 52).complex which could subsequently be oxidized The direct precursor of H2 in anaerobic aminoto "acetyl-coenzyme" and H, (45). acid decompositions is unknown; since the

c. Clostridium aceticum. Several anaerobic clostridia do not manifest formic hydrogenlyaseheterotrophs can effect a total synthesis of acetic activity, it is unlikely that formate is involvedacid from C02, the metabolic hydrogen required as an intermediate. With the exception of am-for this reductive process being derived from monia, the products of amino acid fermentationdegradation of purines, sugars, amino acids are essentially the same as those observed in(glycine), or molecular hydrogen (for detailed decomposition of carbohydrates, and it, there-discussion see 46). The latter is used as the fore, seems probable that H, is formed by thereducing agent by C. aceticum, which grows in a same fundamental reaction in both cases (e.g.,medium containing H2, C02, and unknown through pyruvate breakdown).organic substances present in extracts of mud With certain other species, such as C.(47). Under these circumstances, the principal propionm, single amino acids are decomposedover-all (energy yielding) reaction which to C02, NH,, and volatile acids, but the metabolicoccursis: hydrogen resulting from oxidations is used for

-.e + 22CH,000H + 2H,0 reduction of oxidized intermediates (53, 54). In4H, + 200, --* C~sCOOH + 2H20 these orgasms, there is no coupling with aAlthough C00 also apparently can be the hydrogenase system, and H, is accordingly not

major source of cellular carbon, the necessity produced.for complex supplements categorizes the organism In contrast to the foregoing, many clostridiaas a heterotroph, according to the usual under- (e.g., C. sporogene) do not attack single aminostanding of this term. Karlsson et al. (48) noted acids but instead perform the well known Stick-that H, and C00 are converted to acetate by C. land reaction, in which one amino acid is oxidizedaceticum during growth in a yeast plus malt ex- while another is simultaneously reduced. Usingtract medium and, in addition, grew the or- general terms, this oxidation-reduction reactionganism in a synthetic medium containing glucose, can be represented as:glutamic acid, biotin, pyridoxamine, and panto- R'CHNHECOOH + RCHNHCOOH + H20thenic acid. It has already been noted that (Hydrogen (Hydrogenseveral types of autotrophs utilize H2 for reduc- donor) acceptor)tion of C02 to cell materials. Though themechanism of acetate synthesis from C02 and H2 R'COCOOH + ROHOOOH + 2NH,

is still poorly understood, it is evident that the With C. sporogenes as the test organism, Sticklandoccurrence of this type of reaction in a "hetero- (55) determined which amino acids act as hy-troph" represents an aspect of C00 fixation which drogen donors and which act as acceptors byhas great significance for comparative bio- measuring ammonia production from singlechemistry. amio acids in the presence of the oxidized or

reduced forms of suitable dyes. As expected, theB. Organic Nitroge Compous as Precursors classical Stickland reaction occurs only if one

of the amino acids is from the hydrogen donorMolecular hydrogen appears as a characteristic group and the other from the hydrogen acceptor

end product of amino acid fermentations by group. As already implied, demination of avarious clostridia. Certain species, e.g., C. "hydrogen-acceptor amino acid" occurs in atetanomorphum, can use a single amino acid, system in which the "hydrogen-donor aminosuch as glutamic, as the primary carbon and acid" is replaced by a reduced dye of the propernitrogen source for growth. In addition to molec- redox potential. Similarly, substances such asular hydrogen, C02, NH, and volatile acids glucose and pyruvate can function, in certain(acetic and butyric) are produced during the instances, as reductants for the "hydrogen-decomposition (49). This type of breakdown is 2 For completeness a brief summary of the Stick-not restricted to growth conditions as evidenced land reaction is given here; see article by Nismanby the fact that resting cell suspensions of such in this issue (Bacteriol. Revs., 18, 16-42) for de-bacteria also catalyze qualitatively similar tailed discussion.


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acceptor amino acid" (56). It might be antici- on the other hand, is rapidly and quantitativelypated that amino acids in the hydrogen-acceptor converted to C02 and propionic acid. This ob-category could also be reduced with H2 by servation taken together with the finding thatclostridia which possess the hydrogenase system. conversion of lactate to propionate is dependentC. sporogenes does contain the latter and can, in on a suitable C02 concentration in the mediumfact, link HI oxidation with reduction of acceptor implicates succinate as an intermediate in theamino acids as indicated by the following formation of propionate from Cs compounds byexamples (57).' this organism (62). In this connection, it is of

N1E4CE6COOH + Hs --), NoE4 + CH:COOH interest that the anaerobic dark conversion ofpyruvate to propionic and other fatty acids by

CHNH2(CH,)2CHNH2COOH + HEs Rhodopirumrubrum also shows marked de-(ornithine) pendence on the concentration of C02 in the

NH. + CHNH.(CHE),COOH Stem (64)-(6-minovaleric acid) Except for production of traces of propionic

acid, the decomposition of pyruvate' by V.HjC- --gaogene can be represented by the "phosphoro-

H1c\HcOOH+ H. elastic reaction" typical of C. butylicum (62). Then\k; CHCOOH + He close similarity between the two organisms in thisN respect is further emphasized by the fact thatH neither displays the formic hydrogenlyase reac-

(proline) tion. One point of difference, however, is that theCE,2NHs(CH~shCOOH rate of H, formation from pyruvate by V. gazo-CHNH(CH,)COOH genes is not significantly dminished by an

(8aminovaleric acid)ge isntdmihdby a

atmosphere of H2. In this regard, the organism2. vEiLwNzLUA GAzoGENzs (mcRococcus resembles R. rubrum, in which photochemical

LACTIYCUS); M. AoOsNS H, production is not inhibited by the presenceV. gazogenes is a gram variable strict a

of excess hydrogen (65, 66). Although C.

which physiologically resembles both propionic Iylicum and V. gazogenes differ in this respect,acid bacteria and certain types of the clostridia. it seems likely that the m anism of H2 forma-

tion from pyruvate is essentially the same inThis k--awhich can be readly isolated btboth oreganismsfrom sheep rumen, sheep saliva, human saliva,and other animal sources (60, 61)-is incapable of rSeveralputnes are actively fermented byfermenting sugars and amino acids but vigorously formationellsofHadother proadugcsy () thedecomposes lactate, pyruvate, malate,oxala.e- formation ofHu and other products (67). The

tate, fumarate, tartrate, awl succinate (60, 62).Fermentation of these substances (with the excep- products depend strongly on the particular strain

tion of succinate) is characterized by formation studied. Of particular pertinence to the presentof H2; the presence of a typical hydrogenase in topic, are several hydrogenase linked reactionsthe cells is indicated by the rapid reduction of involving hypoxanthine, and uric acidmethylene blue under an atmosphere of H2 (62). observed in strain 221. Under an atmosphere of

In addition to molecular hydrogen, the other N,, approximately 90 per cent of added hypo-end products observed in lactate fermentation nthine is decomposed to equivalent amounts ofend~~~~~~~~~~~~~~~~~Hproductobsrvem lactate permecentoare C02, acetic and propionic acids (60, 61). xtheandH, whilethe g 10per centTartrate decomposition, which is dependent is converted to 00, onia, and propionic

e~~acid. Under an atmosphere of H2, however, onlyon the presence of appropriate adaptive enzymes,4 acid. Underanathereo however, ol

yiedsheamecomouns (0, 2).Sucinaeabout 40 per cent of the added hypoxanthine ismetabolized, yielding an equivalent quantity of

' It is of interest that Escherichia coli, which xanthine. Since the cells can reduce xanthinedoes not catalyze the Stickland reaction (58), to hypoxanthine with H,, the inhibition of hypo-can reduce methionine sulfoxides with molecular Anthine fermentation by H2 appears to be ahydrogen (59). reversal of the reaction:

4 Fermentation of tartrate by certain clostridiais also adaptive (63). hypoxanthine -. xanthine + H,


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A similar relationship between xanthine and vessel, the glycine decomposition can be approxi-uric acid is suggested by the observations that mated by the equation:uric acid (a) appears as a product of xanthinefermentation under N2 but not under H2, and 4NHs + 2.5CH3COOH + 3CO2 + 1.5H2(b) is reduced to an equivalent amount ofxantbine in the presence of excess H2. It has been Cardon and Barker (54) advanced an explanationsuggested by Whiteley and Douglas (67) that for the absence of H2 as an end product under thethe anaerobic oxidation of hypoxanthine to first set of circumstances based on the observedxanthine and 12 represents a new pathway of H2 effect of H2 pressure on the course of the fermen-formation in microorganisms. Though this may tation. In respirometer experiments using variousbe true, no evidence exists to exclude the possi- N2 plus H2 mixtures, they found that H2 produc-bility that the carrier systems between the tion from glycine was strongly suppressed byhypoxanthine activating enzymes and hydro- the presence of H2 in the gas phase. When thegenase are the same as in other H2 producing H2 content of the atmosphere was greater thanreactions. 25 per cent, no H2 was evolved and, in fact, smallThe reduced purines, hypoxanthine and uptakes of H2 were frequently observed under

adenine, can also be decomposed with the pro- mixtures containing more than 30 per cent H2.duction of H2 by Micrococcua aerogenee (68). This Since 12 is very insoluble in aqueous media, itorganism, however, differs physiologically from was suggested that the formation of very smallM. lacyticu in a number of respects, e.g., it amounts of this gas during the initial stages ofcan ferment certain amino acids but is incapable growth in a system with negligible gas volumeof decomposing lactate. In addition toH2, varying would suffice to raise the H1 pressure to thequantities of C02, ammonia, acetic, propionic, point at which further H2 production is inhibited.and lactic acids result from breakdown of the In a system with relatively large gas space, how-two purines. The pyrimidines, uracil, thymine ever, considerable H2 could be formed beforeand cytosine, are also slowly fermented to the the inhibitory level is reached.same end products. The H2 producing reactions From the foregoing, it is evident that theare apparently not as easily reversible as in M. H2 producing reaction in D. glycinophilus isaciyticus since the purine fermentation patterns readily reversible and that this characteristic isare the same under N2 and H2, and neither important in determining whether metabolicxanthine nor uric acid are reduced in the presence hydrogen is used for reduction of an organicof 12. intermediate or for evolution of H2. Since formate

and pyruvate are not metabolized at appreciable3. DIPLOCOCCUS GLYCINOPHILUS rates by the organism, it appears unlikely that

In comparison with many of the anaerobes these compounds are involved as "direct" H2already discussed, D. glycinophilus has unusually precursors in the glycine fermentation. Therestricted fermentative abilities-glycine and mechanism of acetate formation by D. gly-simple peptides containing glycine with a free cinophiluw seems to be quite complex (69) and is,carboxyl group appear to be the only compounds unfortunately, still poorly understood; it iswhich the organism can decompose (54). A conceivable that H2 is generated, at least in part,rather remarkable characteristic of the glycine by the anaerobic oxidation of a reduced 02fermentation is the pronounced influence of a iteediatephysical factor on the quantitative and qualita- 4. THE miETLkNE BACTERIAtive aspects of the decomposition. Thus, in aclosed system, in which there is negligible gas One of the major mechanisms for methanespace, the fermentation reaction is: formation by microorganisms is represented by

the over-all equation:4NH2CH2COOH + 2H204NHs + 3CH3COOH +2CO2 C02+ 4H2A -- CH4 + 2H20 + 4A

Alternatively, when the inert gas to liquid volume In this type of process, methane carbon is de-ratio is rather large, as in a shaking respirometer rived solely from C02, and the metabolic hydro-


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19541 OXIDATION AND EVOLUTION OF MOLECULAR HYDROGEN 51

gen necessary for the reduction originates from could utilize H2 for reduction of various one-compounds (H2A) such as primary or secondary carbon compounds (CO, C02, HCHO, CH3OH)alcohols, certain fatty acids, or molecular hy- to methane. Subsequent work suggests that thesedrogen (70, 71). C, compounds are not directly reduced toThe formation of methane from C02 and H2 methane, but rather that they are converted

has been conclusively demonstrated in pure through a series of intermediates which may be,culture experiments with Metaiwbacterium in part at least, common with those involved inomelianakii (72, 73), Meth4anosarcina Barkerii' C02 reduction (77). More recently, Schnellen(73, 74), Methanobacterium formiccm (73, 74), (73) and Stadtman and Barker (75) have studiedand Methanmoccu vannielii (75). methane production from formate, with pureM. Barkeii also can convert CO and H2 to cultures of M. formicicum and M. vannielii,

methane (73, 74) according to the reaction: respectively. Since from a metabolic standpointboth organisms appear to be very similar, only

CO + 3H2 --), OH4 + H20 the characteristics of M. vannielii will be con-Carbon monoxide is transformed to methane sidered here. This organism is a strict anaerobeby this organism even in the absence of added which cannot grow on glucose, various commonH2. Under these conditions, i.e., under an at- organic acids or short-chain alcohols. Vigorousmosphere of CO and N2, considerable amounts of growth occurs in an alkaline mineral plus formateC02 are observed as a product of the "fermenta- medium (containing Na2CO3), with the concomi-tion". Furthermore, when this "fermentation" tant formation of H2, C02, and methane. Duringproceeds in the presence of a C02 absorber the early stages of growth, formate appears to be(alkali), appreciable amounts of H2 accumulate decomposed to C02 and methane; coincident within the gas phase. These results are consistent increasing alkalinity of the medium (beyond pHwith the following general mechanism: 8.6), conversion of formate to C02 and H2 be-

4CO + 4H20 - 4CO2 + 4H2 comes the major reaction.C02 + 4H2 -+ CH4 + 2H20 Formate is anaerobically decomposed to

equimolar amounts of H2 and C02 by resting4C0 + 2H20 > 3002 + OH4R cell suspeons of M. vannielii at an initial pH

of 8.1. Such suspensions (initial pH 6.8-8.1)Thus, CO first undergoes a reaction in which C02 also consume H2 rapidly in the presence of C02and H2 are produced, and methane is subse- with the production of both formate andmethane.quently formed from these products according The apparent ease of reversibility of the reaction:to the generalized equation given previously. It HCOOH - H2 + CO2 in this organism suggestsis of interest that M. Barklei catalyzes the the probability that the cells might effect aover-all reaction even under an atmosphere of rapid exchange of carbon between C02 and100 per cent CO. M. formicicum effects a similar HC1400H. Stadtman and Barker (75) testedconversion, but only at lower partial pressures of this, and although interpretation of their resultsCO. It was already noted that M. omelianskii is complicated by the simultaneous net decom-produces methane from C02 and H2-this bac- position of formate to C02 and H2, the dataterium, however, is inactive with regard to CO, clearly indicate exchange between formate andwhich provides additional evidence indicating C02. It was reported (75) that dried cell prepara-that the latter compound is not an intermediate tions of M. omeliansuii also catalyze the exchange.in methane formation from C02 (74). These results emphasize a fundamental differ-

Several investigators observed the transforma- ence between the anaerobic degradation oftion of formate to methane many years ago using formate to C02 + H2 and the aerobic conversioneither crude enrichment cultures or other cultures of forMate to C02 and water The experimentof doubtful purity. In more recent times, Stephen- of Da ttson(78) and of Mathews and Venneslandson and Stickland (76) isolated an unidentified (7 8)an the wsan stadorgaismprbablinpur cuture whch ro- (79) show that the latter process, as studied

ducedism,methan yfo forme and which alo- with partially purified formic dehydrogenase

preparations from higher plants, is for all prac-5 Name in original paper. tical purposes irreversible and that there is no


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significant exchange of labeled carbon between hydrogenase; and second, it was observed that01402 and formate in the presence of the enzyme. certain other orisms displayed both formicWith reference to the methane fermentation, it dehydrogenase and hydrogenase activities butis evident that the occurrence of a rapid exchange not hydrogenlyase. The two-enzyme hypothesisreaction in M. vannielii disqualfes simple tracer was consequently abandoned, and insteadexperiments as a suitable means of determining "formic hydrogenlyase" was considered to be awhether formate is directly reduced to methane distinct single enzyme. Subsequent work byor, alternatively, whether formate is first con- Ordal and Halvorson (86), however, showed con-verted to C02, which is subsequently reduced to clusively that hydrogenase, as measured bythe hydrocarbon. In conclusion, it is pertinent reduction of methylene blue under H2, was pres-to note that the intermediates in reduction of ent in the three strains of Aerobader aerogenesC02 and other C1 compounds to methane may and the two strains of A. cloacae which theywell be multicarbon compounds and, conse- tested (86). With regard to the second point, it isquently, the simple equations used above must evident that the absence of hydrogenlyase ac-be regarded as over-all reactions (77). tivity in an organism containing formic dehydro-

genase and hydrogenase is not necessarily con-5. THIE COLFON-AEROGENES BACTERIA tradictory to the two-enzyme theory since it is

Various organisms of the colon-aerogenes group possible, as indicated by Ordal and Halvorson,characteristically produce large amounts of H2 that an esetial factor required in addition toduring anaerobic metabolism of carbohydrates. the two enzymes may be lacking.The work of Pakes and Jollyman (80) and of Further support for the two-enzyme conceptHarden (81) at the turn of the century strongly is found in comparative studies on the distribu-implicated formic acid as the immediate hydrogen tion of formic dehydrogenase and hydrogenaseprecursor in these degradations. This conclusion in normal and nongas producing variants ofwas based on three important observations: (a) Ewherieia coli (86). The "anaerogenic" variantsadded formate is converted to C02 and H2 by generally lack either hydrogenate, formic de-gas producing strains; (b) nongas producing hydrogenase (estimated by methylene blue re-genera or strains do not catalyze this conversion; duction), or both enzymes. Some variants containand (c) formic acid accumulates as a major both enzymes but, as indicated above, may beend product in carbohydrate fermentations by devoid of a third essential factor. Furthermore,the latter type of organism (see also 82). formic dehydrogenase and hydrogenase areThe decomposition of formate according to always found in cultures which have reverted to

the reaction: HCOOH v H2 + C02, is attributed the gas producing form. Evidence of a more directto the activity of an enzyme system designated nature for the multienzyme character of hydro-as "formic hydrogenlyase" (83). In 1931, genlyase is provided by studies with cell-freeStephenson and Stickland (84) suggested that systems (see later). Recognition of this enzymatic"hydrogenlyase" was actually a two-enzyme complexity is essential for a more meaningfulsystem in which formic dehydrogenase and appreciation of the effects of various nutritional,,drogenase act in conjunction, i~e., factors on the development of hydrogenlyase

activity in the colon-aerogenes organisms. In

HCOOHformic

C0 + 2H+ + 2e1937, Stephenson (85) summarized the available

dehydrogenase data and concluded that at least four factors

hydrogenase H "operating during growth affect the occurrence2H+ + 2e H2 of formic hydrogenlyase in washed suspensions

hydrogenlyase of organisms of the Bacteriaceae:-1. the degreeHCOOH i- -Y C02 + H, of aeration, 2. the presence of formate, 3. the

This postulated mechanism was soon discarded presence of hexose or some other substance, e.g.glycerol or pyruvic acid, which on fermentation

by its proponents for two reasons (83, 85). First, gives rise to formate, 4. some unknown constituentit was found that four strains of Aerobacter of broth. (With respect to 4, one must remember(Bacterium lctis-aerogene) possessed hydro- that a number of amino acids when oxidized bygenlyase activity but appeared to lack the enzyme bacterial action give rise to formic acid)."


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These factors, as well as some others subsequently to specify the locus of action of oxygen on thefound to be of importance, are discussed in the system since several of the components are in-following sections. hibited by this agent to different extents. Pre-

formed hydrogenase is inactivated by oxygen (seeA. Influence of Nutiol Fars on H section II. 5,D). Similarly, the activities of the

Mekzbolism soluble (90) and particulate (87) formic dehydro-a. Oxygen. Formic hydrogenlyase activity is genases are adversely affected by 02. In addition,

absent in E. coli when the organism is grown it is possible that an intermediate factor operat-with vigorous aeration. Cells cultivated in this ing between the two enzymes is inactivated bymanner contain a high level of formic dehydro- oxidation.genase (87, 88) and, in the experience of the The absence of H2 producing systs inauthor, show either little or no hydrogenase aerobically grown organisms has, of course, anactivity.' Several investigators (e.g., 89), how- important bearing on the end products ofever, have reported the presence of hydrogenase anaerobic carbohydrate metabolism in such cells.in intact or cell-free preparations of "aerobically- It was noted previously that formic acid accumu-grown" cells. It appears then that hydrogenase lates in carbohydrate fermentations by nongscan be formed, to some extent at least, when producing variants. In contrast to anaerobicallymoderate aeration rates are employed, but that grown (normal) cells which metabolize pyruvatewith sufficientlyhigh rates the enzyme is virtually with the formation of H2 and C0,, cells grownabsent. The dramatic effect of oxygenation on the with aeration 4ecompose this intermediate fer-synthesis of hydrogenase (or of an accessory Mentatively by the dismutation and the classicalenzyme or cofactor) is clearly reflected by com- phosphoroclastic reactions (94).parative activity assays of extracts and b. Formats and fermentabl carbohydrates.particulate preparations from cells grown semi- Formation of the hydrogenlyase complex duringanaerobically and with vigorous aeration (90). anaerobic growth is dependent on the presence ofReports conflict concerning the effect of oxygen formate or of a ready source of formate, such as

on the already formed hydrogenlyase complex fermentable carbohydrate (83, 85, 95). Hence,present in anaerobically cultivated cells. Stephen- one or more of the components of the systemson and Stickland (83), Yudkin (91), and Pinsky is presumably an adaptive enzyme. This is clearlyand Stokes (92) indicate that aeration has little indicated by the work of Stephenson and Stick-or no effect on the activity of the system. Lalls land (95), who found that suspensions of E. coli(93), on the other hand, found that hydrogenlyase (containing no apparent hydrogenlyase) could beactivity was strongly depressed by exposure to induced to form the enzyme complex by anaerobicoxygen and that the lost activity could be largely incubation with formate in a phosphate bufferrestored by anaerobic incubation of the aerated plus tryptic broth mixture. The activity appearedcells with traces of fermentable carbohydrates. with the kinetics characteristic of an adaptive(The nature of this reactivation is discussed in response-without appreciable concomitant cellSection II. 5,B.) It seems likely that here again division. It now seems that fermentable carbo-the divergent results are due to varying degrees hydrate (eg., glucose or pyruvate) is importantof aeration and possibly also to strain differences. not only as a reservoir of formate, but also as aThe hydrogenlyase activity in cell-free extracts source of: (a) energy required for enzyme syn-of E. coli shows a definite sensitivity to oxygen. thesis; and (b) an integral "coenzyme"When such preparations are shaken in contact component of the complex (see later). The adap-with air for several hours, formate decomposition tive formation of hydrogenlyase in nonproliferat-occurs only after a prolonged induction phase ing aerobically grown cells of E. coli thus requires(90). The latter is markedly shortened by addi- in addition to formate, fermentable carbohydratetion of catalytic quantities of a low redox po- and also certain amino acids which presumablytential dye such as methyl viologen, but not by function as protein precursors (92). Adaptationcompounds such as homocysteine. It is difficult in the presence of these supplements occurs more

' Unless noted otherwise, hydrogenase activity readily in stationary phase cells than in youngis defined on the basis of H, utilization with meth- cells (96).ylene blue as acceptor. It is significant that the level of formic dehydro-


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54 HOWARD GEST [YOL. 18

genase activity remains the same during the are also stimulatory (e.g., tyrosine plus cystine).adaptation (of aerobically grown cells), whereas Incubation of cells obtained from the syntheticthe hydrogenase activity rises markedly. Al- basal medium with a mixture of glucose andthough Pinsky and Stokes (92) regard the in- glutamate for several hours leads to appearancecrease in hydrogenase level as "an incidental of hydrogenlyase activity in the absence of ap-adaptation to a product of formic hydrogenlyase preciable cell multiplication. As already noted,activity", it seems more likely that this increase Pinsky and Stokes (92) found similar require-is one of the essential requirements for developments for the adaptive formation of hydro-ment of the active complex. Some of the argu- genlyase in aerobically grown cells. The aminoments advanced by Pinsky and Stokes in this acid requirements for adaptation in cells grownregard are based on quantitative comparisons of with aeration appear to be more stringent sinceformic dehydrogenase, hydrogenase, and hydro. appreciable enzyme formation was observed onlygenlyase activities. It must be emphasized that when arginine and aspartic acid were employedthe two former enzymes are usually estimated in addition to glutamate. Since amino acidsusing artificial electron acceptors such as have no effect on the activity of the preformedmethylene blue. Further, the assay of hydro- hydrogenlyase system in intact cells, it is evidentgenase is, of necessity, based on measurement of that these compounds are required for proteinH2 utiliation rather than evolution. This being synthesis. Kushner and Quastel (99) recentlyso, quantitative comparisons with the activity of also have studied the amino acid requirementsthe "natural" H2 producing system (hydro- for hydrogenlyase formation, and their resultsgenlyase) are not always justified and may are in essential agreement with those describedactually be quite misleading. Unfortunately, this above.important aspect of the multienzyme versus Cells from a synthetic glucose medium possesssingle enzyme controversy has not been generally formic dehydrogenase, and, as measured byappreciated. In contrast to the experiments with Billen and Lichstein (98), supplementation ofE. coli described previously, Lichstein and Boyd the medium with amino acids increases the level(97) have recently reported that aerobically of this enzyme but not nearly as much as observedgrown cells of A. aerogenes D-1, exposed to with the hydrogenlyase system. The formichydrolyzed yeast extract and formate, showed dehydrogenase activity was estimated by measur-hydrogenlyase activity after a lag period but that ing C02 production in the presence of methylenethe adapted cells did not reduce methylene blue blue. Although this may be an adequate assaywith H2. These investigators assumed that under certain conditions, it cannot be con-hydrogenase was absent and concluded that sidered as a specific method for formic dehydro-hydrogenlyase is a distinct single enzyme. Since genase in the present case since the rate of C02ability to reduce methylene blue is an arbitrary production is here a measure not only of theindex of hydrogenase activity, this interpretation assumed "direct" transfer of electrons tocannot be accepted without supplementary tests methylene blue (MB), but also possibly of thefor hydrogenase using other criteria (e.g., ability activity of the over-all hydrogenlyase system.to catalyze the exchange reaction or to reduce Thus when hydrogenlyase activity is present, theother dyes). following may occur.

c. Amino acids and ammonia. During a study HCOOH C002 + H2of the various factors concerned with formation H2 + AM MBH2of the hydrogenlyase complex, Yudkin (91)found that cells of E. coli from a mineral salt HCOOH + MB C02 + MBH2plus glucose medium were unable to decompose The over-all reaction is, of course, the same asformate to C02 and H2. The subsequent investi- would be observed if only formic dehydrogenasegations of Billen and Lichstein (98) showed that were present.the active hydrogenlyase complex is present in When formate is decomposed by the hydro-E. coli when the simple synthetic medium is genlyase system in the presence of methyleneaugmented with amino acid supplements. The blue, H2 is not evolved (90). In view of this fact,most effective single amino acid is glutamic, it is difficult to interpret the experiments ofwhile certain combinations of other amino acids Grunberg-Manago et al. (100) who reported that


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low concentrations of sodium hypophosphite below about 12 per cent (of the dry weight).inhibit formic dehydrogenase activity (measured Both activities are essentially zero at a nitrogenby methylene blue reduction) completely but content of approximately 10.7 per cent. Thehave no effect on the hydrogenase or hydro- "formic dehydrogenase" activity, on the othergenlyase activities in intact cells of E. coli. hand, actually increases with nitrogen deficiencyThese authors conclude that hydrogenlyase is a over this range. These results indicate thatsingle enzyme not related to formic dehydro- hydrogenase is a necessary component of thegenase or hydrogenase. If this were so, it would hydrogenlyase complex and are interpreted byseem that in the presence of the "formic dehydro- DeLey as indirect evidence in favor of the two-genase" inhibitor, the hydrogenlyase reaction enzyme hypothesis. Virtanen and co-workerswould occur and that, as a consequence, (103, 104) believe that under conditions ofmethylene blue should have been reduced ac- limited supply, the available nitrogen is prefer-cording to the foregoing equations. This entially used for synthesis of constitutive ("in-contradiction cannot be satisfactorily explained dispensable") enzymes and, accordingly, thatat present, but it should be noted that these adaptive ("dispensable") enzymes are notobservations do not exclude "formic dehydro- elaborated under these circumstances. Conse-genase" as a component of the hydrogenlyase quently, hydrogenase would be regarded as ancomplex; it is conceivable that hypophosphite adaptive enzyme which is not essential for cellsomehow inhibits electron transport to the dye multiplication. The requirement for amino acidbut not to the "natural" electron acceptor of supplements in the production of hydrogenaseformic dehydrogenase. (or hydrogenlyase) by the other strains of E. coli

It is probable that the inability of organisms discussed above could of course be interpretedfrom a mineral salt plus glucose medium to de- on a similar basis.compose formate by the hydrogenlyase mecha- d. Nitrate and fumarate. The inhibitory effectnism is in large part due to the virtual absence of of nitrate on the hydrogenlyase system in colon-hydrogenase in such cells. The level of hydro- aerogenes organism was first observed by Pakesgenase activity with both methylene blue and and Jollyman (105), who noted that H, was notfumarate is greatly increased by addition of produced in formate or glucose broth culturesamino-acids, particularly glutanate and methio- containing one per cent KNOs. Suspensions ofnine, to the growth medium (101). Incubation of resting cells obtained from such media are in-nonproliferating cells with glutamate under the capable of producing H, from formate and glucoseproper conditions similarly leads to appearance (91) which suggests that nitrate actually inhibitsof hydrogenase activity (98). From these inves- synthesis of one or more enzymes of the hydro-tigations it is evident that development of genlyase complex. The experiments of Billenhydrogenlyase activity is paralleled by develop- (106) indicate that hydrogenase synthesis isment of hydrogenase activity, which is further markediy suppressed by inclusion of 10-20 mgsupport for the two-enzyme concept. of anmonium nitrate per 100 ml of medium,The strain of E. coli used by DeLey (102) whereas these quantities of the nitrate appear

seems to differ from those used by other investi- to stimulate formic dehydrogenase formation.gators in that cells from a mineral salt plus su- The presence of adequate levels of casein hy-crose medium show hydrogenlyase activity, drolyzate in the medium counteracts to a largeprovided that the ammonium sulfate content of degree the inhibitory effect of nitrate on thethe medium is sufficiently high. DeLey made appearance of hydrogenase and hydrogenlyasecomparative assays of hydrogenlyase, hydro- activities. Billen therefore suggests that nitrategenase, and "formic dehydrogenase" in cells of may act by stimulating formation of the adaptivedifferent nitrogen content, obtained by growth enzyme nitratase thereby depleting the pool ofin media containing varying amounts of am- nitrogenous precursors also necessary for synthe-monium sulfate. When enzyme activity is plotted sis of enzymes of the hydrogenlyase complex.as a function of decreasing nitrogen content of Nitrate inhibits adaptive formation of hydro-the cells, the results show that hydrogenase and genlyase in aerobically grown cells (92) and alsohydrogenlyase activities begin to fall markedly the activity of the preformed system in intactwhen the nitrogen content of the cells diminishes organisms (83) and in cell-free extracts (90).


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The latter observations indicate a direct effect would be evident. In glucose plus amino acids,of nitrate (or its reduction products) on one or on the other hand, energy for protein synthesis ismore enzymes of the complex, and it appears, derived from glycolysis, which is of course inde-therefore, that the action of nitrate cannot be pendent of the presence of fumarate; accordingly,explained solely on the basis of diversion of pro- ammonia would not inhibit under these condi-tein synthesis in favor of nitratase formation. tions. Furthermore, the inhibitory effect of am-

E. coli and other colon-aerogenes organisms monium chloride in peptone plus formate wasactively reduce nitrate to nitrite and thence to shown to be counteracted by addition of nitrate.ammonia with organic compounds or H2 serving Since the latter could function as a hydrogenas reductants. Nitrite effectively inhibits adaptive acceptor in lieu of fumarate, this result is con-hydrogenlyase formation in "aerobic" cells (92) sistent with the proposed explanation. To recon-and activity of the preformed system (83). cile these effects of nitrate with the resultsConsequently, the inhibitions observed with reported by others, Kushner and Quastel suggestnitrate may actually be due to nitrite, produced that the energy contribution resulting fromthrough the action of nitratase. Depending on oxidation of formate with nitrate as acceptorthe levels of nitratase and nitrite-reductase may, in some cases, overcompensate for thepresent, varying degrees of inhibition by nitrate inhibitory effects of this anion and that oneor nitrite may be obtained. The exact locus of might reasonably expect the "balance" betweenaction of nitrite on the preformed hydrogenlyase these opposing effects to vary from strain tosystem is still unknown. Apparently, marked strain. Although no evidence is available, itstrain differences exist in E. coli with regard to appears that the strains used in their investiga-the effect of nitrate on adaptive formation of tion must have an unusually high nitrite-hydrogenlyase in nonproliferating aerobically reductase activity, which would be required forgrown cells. Kushner and Quastel (99) found with rapid removal of the toxic nitrite produced bytheir particular strains that the final QH, values nitrate reduction.attained after adaptation in the presence of e. Metals. The importance of metals in hydro-formate and peptone were considerably greater genlyase activity has been established in studiesif nitrate, fumarate, or aspartate were also added with intact cells of Aerobacter indologenes (107)to the incubation fluid. The hydrogenlyase ac- and with the cell-free enzyme system fromtivity of preadapted cells was not stimulated by E. coli (90, 108).the latter compounds, and, in fact, nitrate and In contrast to the fermentation pattern offumarate depressed H2 evolution, no doubt be- normal organisms, anaerobic decomposition ofcause they functioned as hydrogen acceptors. glucose by iron deficient cells of A. indologenesAccording to Kushner and Quastel, nitrate is characterized by absence of H2 as a product

and fumarate enhance the adaptive process by and by accumulation of large amounts of formicacting as hydrogen acceptors for oxidations (of (and lactic) acid. Thus, the ability of this or-formate or peptone ingredients) which provide ganism to produce H2 is strongly dependent onthe energy required for enzyme synthesis. iron nutrition. It will be recalled that a similarAspartic acid was presumably effective because dependence was found in the clostridia, and, con-it gives rise to fumarate through the action of sequently, iron seems to have an important roleaspartase (i.e., aspartic *-, fumarate + NHI). in at least two ostensibly different types of H2High concentrations of ammonium chloride production. As might be expected, the catalase(0.1 M) strongly inhibited hydrogenlyase synthe- and peroxidase contents of iron deficient cells ofsis in formate plus peptone but not in glucose A. indloWenes are less than 5 per cent of theplus amino acid mixtures. These observations normal values. Although visual spectroscopicwere explained as follows: In formate plus observations indicate that the cytochromepeptone, fumarate which is required as a content is also appreciably reduced, this may nothydrogen acceptor for energy yielding oxidation be true since the deficient cells show practicallyis generated from aspartic acid present in peptone normal 02 consumption with glucose, and thisingredients. Ammonia in high concentrations respiration is severely inhibited by cyanide.would shift the aspartase equilibrium so that Comparison of normal and deficient cells withlittle fumarate is available and hence an inhibition regard to hydrogenase, formic dehydrogenase,


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and hydrogenlyase activities shows that all these f. Other factors. Lichstein and Boyd (109, 110,are markedly suppressed by iron deficiency. The 111) have implicated oleic and other long-chaineffects of iron depletion cannot be attributed to fatty acids in formate breakdown by colon-a general depression of metabolism since oxidative aerogenes bacteria. The hydrogenlyase activityglucose breakdown and the activities of various of a biotinless E. coli mutant was found to bedehydrogenases in the deficient cells are at the considerably greater in cells obtained from anormal (or only moderately reduced) level. formate + casein hydrolyzate + oleate medium

Consideration of these results by Waring and than in orgaisms from a medium in which oleateWerkman (107) led to the suggestion that there was replaced by biotin (109). This was also trueis an electron carrier between formic dehydro- for the relative formic dehydrogenase activities,genase and hydrogenase which contains func- the measurement of which is subject to thetional iron "probably in a manner similar to criticisms noted previously. In general, similarcytochrome". In view of the fact that the cell-free results were observed with various wild typehydrogenlyase system is strongly inhibited by strains of E. coli and A. aerogenes. Small amountsmetal ion completing agents such as 8-hydroxy- of oleate also caused a striking (and immediate)quinoline and a,a'-dipyridyl, it seems unlikely stimulation of hydrogenlyase activity in restingthat a "cytochrome-like" component is involved suspensions of the mutant grown in the basal(90). Inhibition by these agents is completely (formate + casein hydrolyzate) + biotinrelieved by Fe-+, partially by Mn++, whereas medium. Biotin likewise stimulated the restingother divalent cations are without effect (108). cells but to a considerably smaller degree. OnPartial resolution of the metal activator can be the basis of these results, Lichstein and Boydachieved by precipitating the complex with believe that oleate, or a substance derived fromammoniumsulfate; with such preparations, Mn" this compound, functions as a cofactor in theappears to be somewhat more effective than Fe++ hydrogenlyase system.(90). The requirements for a dissociable metal The oleate effect is not specific since a varietyion cofactor can adequately account for the of long-chain saturated and unsaturated fattyinhibitions observed with cyanide (83, 90) and acids frequently enhance hydrogenlyase activityCO (83). in resting cells of the mutant (110). Since oleicWaring and Werkman dispute the claim that and the other acids do not accelerate the activity

an active hydrogenlyase system cannot be of dried cell preparations of A. aerogenes (111),formed by (Aerobacter) cells grown with con- it seems possible that these compounds may causetinuous aeration. They report that organisms the stimulations noted by altering permeability.grown in a relatively iron rich medium with In this connection it may be remarked thataeration show the same hydrogenlyase activity oleate has no effect on the activity of the dialyzedas cells cultivated under strict anaerobiosis or ammonium sulfate precipitated cell-freeand suggest that with insufficient amounts of system (90).iron, hydrogenlyase may not be formed aerobically Complex materials such as yeast and liver ex-because the metal is preferentially used for tracts stimulate formate decomposition by thesynthesis of more essential metallo-enzymes biotinless E. coli mutant and by dried A. aerogenes(e.g., cytochromes). This explanation implies preparations, particularly after acid hydrolysisthat the media used by other investigators were (110, 111). It was suggested that the active sub-relatively iron deficient, which seems unlikely stance(s) in these complex supplements is derivedif we consider the facts that the iron requirement from a fatty acid precursor, possibly oleic acid.for maximal growth is quite small, and that it The recent work of Broquist and Kohler (112),was undoubtedly met adequately by iron con- however, indicates that the effects of yeasttamination in the inorganic chemicals and com- extract, etc. are due to traces of carbohydrate,plex supplements (yeast extract, peptone) gen- which as shown by the experiments of Lascelleserally employed. Although it appears that (93) can be fermented to a "factor" which mark-"vigorous" aeration was used by Waring and edly accelerates the hydrogenlyase activity ofWerkman, it seems possible that the rate of dilute or aged resting cell suspensions.oxygenation was still insufficient to attain the B. Mechanism of the Hydrogenlyase Reactioninhibitory level. Several aspects of the mechanism of hydro-


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genlyase action have been clarified by recent proportional decline over the same dilutionstudies with cell-free preparations from anaerobi- range (90). These observations hold for the ex-cally grown E. coli (45, 90, 113, 114). Concen- perimental arrangement in which the extract istrated extracts obtained by grinding with diluted with buffer, and formate added from thealumin display hydrogenase, "formic dehydro- side arm of the Warburg vessel at zero time. Itgenase",7 and hydrogenlyase activities when has been observed, however, that a concentrationtested at pH 6.0-6.2 in phosphate buffer. Hydro- of extract which shows no hydrogenlyase activitygenlyase activity is im in this pH range and in such a test frequently does display activity ifdiminishes to a negligible value if the pH is in- the enzyme aliquot is kept undiluted in the sidecreased to 7.2 or higher. Intact cells, on the other arm and added at zero time to the formate plushand, generally can decompose formate to H2 and buffer mixture (Swim, H. E., and Gest, H.,CO2 in the neutral or alkaline range (83, 93), unpublished data). Control tests with reducingbut the optimal pH varies depending on the agents indicate that the lack of activity underformate concentration (85, 115). Although the the first set of circumstances is not due to in-soluble cell-free system shows no hydrogenlyase activation, during manipulations, of an enzymeactivity at pH 7.2, high levels of hydrogenase and component by traces of oxygen dissolved in theformic dehydrogenase activities are observed liquid (45). In experiments wherein the dilutionunder these conditions. Thus at alkaline pH the of extract is insufficient to abolish hydrogenlyasesoluble complex resembles the intact cell prepara- activity completely, gas evolution from formatetions discussed earlier, which possess hydrogenase frequently begins after an induction period ofand formic dehydrogenase but no hydrogenlyase. variable length and continues with kineticsIt would appear that an intermediate reaction in typical of an autocatalytic reaction (90). Thesethe hydrogenlyase mechanism is quite sensitive observations suggest that the effect of dilutionto alkaline conditions. is complex, and that it may involve, at least inAttempts to demonstrate "direct" anaerobic part, dissociation of a cofactor below a critical

oxidation of formate at pH 7.2-7.5 with ac- level, particularly in the absence of formate.ceptors other than methylene blue, e.g., fumarate, In spite of the fact that the explanation of theby both soluble and particulate fractions from dilution effect is not clear, experiments using theE. coli have thus far given negative results. "first method", i.e., with diluted extracts showingThe dismutation between formate and fumarate, only hydrogenate activity, have been of con-first described by Krebs who used suspensions siderable value.of E. coli containing hydrogenlyase, appears to The hydrogenlyase system has been "recon-occur through a combination of reactions structed" by combining "dilute" extract from(90, 116), viz., anaerobically grown cells and a particulate

fraction from aerobically cultivated E. coliH2COOH COa + He (90). The particles obtained from the latter typeH2 + fumarate -. succinate of cell contain cytochromes at and b1 and also

HCOOH + fumarate CO2 + succinate show strong formic dehydrogenase activity;hydrogenlyase and hydrogenase activities are

The available data, however, do not exclude the either absent or at an extremely low level. Inpossibility that in the intact cell formate and the original interpretation of this "reconstruc-fumarate can also interact by a more direct tion", it was considered that the "aerobic parti-route. cles" furnished the formic dehydrogenaseOne of the most outstanding properties of the component, while the "anaerobic extract" pro-

soluble system is the disproportionate effect of vided hydrogenase and possibly intermediatedilution on the hydrogenlyase and formic de- factors. This interpretation tacitly assumed thathydrogenase (tested at pH 7.5) activities. When the lack of activity in the dilute "anaerobicthe concentration of extract is reduced below a extract" was due to dilution of formic dehydro-certain point, these activities show a precipitous genase below a critical concentration. In view offall. The hydrogenase activity, however, displays the foregoing discussion, it appears that this

7 Defined on the basis of C02 production in the assumption may not be justified; although thepresence of methylene blue. particles no doubt supply an additional


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allotment of formic dehydrogenase to the that it is necessary for a reaction in which ansystem, they probably also provide a mechanism essential cofactor is generated.for coupling between the formic dehydrogenase Lcelles (93) has observed that the hydro-and hydrogenase present in the "anaerobic genlyase activity of intact cells in borate bufferextract" fraction. is measurably enhanced by addition of phosphate.

Particulate preparations from anaerobically The activity of the ammonium sulfate precipi-grown cells differ from their "aerobic" counter- tated complex in maleate buffer is similarlyparts in a number of respects (90). The former stimulated by phosphate, but this may be ancontain formic dehydrogenase, cytochrome b1 indirect effect concerned with stability of the(but not a2), and show excellent hydrogenase system (90). Decomposition of formate by theactivity with methylene blue, ferricyanide, precipitated complex in phosphate buffer isnitrate, and fuxmarate. When fresh, the particles inhibited by arsenate in a noncompetitiveusually show hydrogenlyase activity after a manner, reminiscent of the effects of arsenate onnoticeable induction period. The induction phase the fixation of formate into pyruvate (117).is almost completely eliminated when catalytic C2 derivaties. After anaerobic dialysis againstamounts of low redox potential dyes (e.g., methyl water, free of dissolved oxygen, the "concen-or benzyl viologen) are present. These dyes trated" extract from E. coli is incapable ofpresumably can act, under certain conditions, decomposing formate or does so only after aas electron carriers between formic dehydro- prolonged induction period (90). This inductiongenase and hydrogenase. They cannot com- phase is markedy shortened or abolished whenpletely substitute for the "natural" pathway the dialyzed extract is preincubated with boiledsince aged particles, which contain active hydro- preparations from E. coli, C. butylicum, yeast,genase and formic dehydrogenase but no longer and pigeon liver. Among the variety of purifiedexhibit delayed hydrogenlyase activity, do not potential cofactors tested, only pyruvate, di-produce gas from formate upon addition of acetyl, and acetyl phosphate were found toviologen dyes. Hydrogenlyase activity is ob- "spark" formate decomposition by dialyzedserved, however, after an induction period when extracts. These compounds are individuallythe aged particles are combined with "dilute" capable of activating the reaction, generally afterextract. Except for the presence of hydrogenase an induction period (45). Acetyl-coenzyme A,in the particles, this "reconstructed" system on the other hand, appears to be incapable ofsuperficially appears to be essentially equivalent "sparking", and it has also not been possibleto the one discussed previously. Insufficient to demonstrate a coenzyme A requirement fordata are available to warrant further speculation hydrogenlyase activity. As in the "dilute"on the exact nature of the unquestionably com- enzyme experiments, the "concentrated" dialyzedplicated synergistic relationships between extract frequently shows appreciable immediate"dilute" extracts and particles of anaerobic activity when it is added from the side arm oforigin. The particulate fraction seems to be the Warburg vessel to the formate plus buffersolubilized to some extent by sonic vibration, mixture at zero time'(Swim, H. E., and Gest, H.,and it is likely that further study of such prepara- unpublished data). Evidently the effects oftions will facilitate an understanding of these dialysis cannot be completely explained as dueinteractions. to total depletion of a dissociable cofactor.

In addition to the metal ion cofactor (see II. Under proper conditions, small quantities of5,e), studies with the soluble complex have pyruvate, diacetyl, or acetyl phosphate are alsoindicated roles for cocarboxylase, phosphate (?), capable of "sparking" decomposition of formateand C2 derivatives in the hydrogenlyase mecha- by the "dilute" (inactive) E. coi preparation,nism (45, 90). The complex precipitated by but only in the presence of C. butylicum extractalkaline ammonium sulfate shows no activity in (45, 114). The Clotridium extract, which doesthe absence of supplements. Addition of Mn++ not metabolize formate, appears to be requiredalone has no effect, whereas activity is observed for converting the added "cofactors" to the(after an induction phase) when Mn++ and co- actual "sparking" intermediate. All of the fore-carboxylase are supplied. It is possible that going observations strongly suggest the partici-cocarboxylase is not directly involved, but rather pation of C2 derivatives in the degradation of


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DIACETYLPYRUVATE ORACETYL PHOSPHATE

FORMATE + ACETYL () [C3]

-2HL ACETALDEHYDE -X + CO2

HpSCHEME A= REACTIONS 1,2,3SCHEME B = REACTIONS la, 3

Figure 1. Possible mechanisms of formate decomposition to H2 and C02. Reproduced from Barkulisand Gest (45) through the courtesy of the Elsevier Publishing Co., New York and Amsterdam.

formate to CO2 and H2; on the basis of the through reversal of reaction 1. Finally the pre-available facts, Barkulis and Gest (45) have sumed absence of reaction 1 in C. butylicumproposed two alternative mechanisms for the could explain the inability of this organism tohydrogenlyase reaction, both employing C2 decompose formate to C02 and H2.derivatives as electron carriers. (Figure 1). Scheme B postulates a "direct" dehydrogena-

According to scheme A (reactions 1, 2, 3), tion (decarboxylation) of formate with acetyl Xformate and an acetyl derivative are condensed serving as the electron acceptor (reaction la).to produce a C3 compound, which is then de- Regeneration of acetyl.X and production ofcarboxylated yielding C02 and an acetaldehyde H2 then occur through reaction 3, as in scheme A.complex. The acetaldehyde compound is oxidized All of the reactions indicated are assumed to beby transfer of electrons to the hydrogenase sys- reversible since the work of Woods (118) withtem, thereby leading to formation of H2 and intact cells of E. coli has conclusively establishedregeneration of acetyl-X. This scheme implies the reversibility of formate decomposition tothat pyruvate decomposition to C02 and H2 by C02 and H2. In connection with reaction 3, inE. coli and C. butylicum may occur through which acetaldehyde-X is oxidized to acetyl.X,the same pathway, viz., pyruvate -+ Ca2kCO2 Barkulis and Gest (45) have suggested that+ acetaldehyde .X-4 H2 + acetyl -X -) acetate. transfer of electrons to the hydrogenase systemIf this were the case, it would be expected that may not be obligatory. Thus, other oxidantsE. coli might, under certain conditions, produce (e.g., methylene blue) could substitute for theH2 from pyruvate (or glucose) but not from hydrogenase pathway, and molecular hydrogenformate. There is some (disputed) evidence that would then not be produced. Accordingly, theE. coli, grown in a particular manner, shows this rate of C02 production from formate in thebehavior [see discussion in (85) and (86) ]. Resting presence of external electron acceptors would becells of colon-aerogenes bacteria obtained by interpreted as an assay of the "formic dehydro-aerobic growth in certain media decompose genase" activity.pyruvate without gas formation by means of It is apparent that identification of the C2the phosphoroclastic reaction. This could be compounds and further evidence which will per-accounted for by assuming that reactions 2 and mit a choice between schemes A and B are re-3 do not take place in such cells and that, al- quired for a better understanding of theternatively, pyruvate is metabolized mainly mechanism of the reaction. In any event, the


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implication of C2 derivatives in hydrogenlyase exchange process but also to evolution of non-activity indicates that carbohydrates are im- isotopic H2. Production of H2 from formate isportant not only as a source of formate and of not completely inhibited by an atmosphere ofenergy for synthesis of enzymes of the system hydrogen, as suggested by Hoberman and Ritten-during growth or adaptation, but also as pre- berg-the rate is only depressed about 40 percursors of an essential "C2-coenzyme". The ob- cent under these conditions (85, 90). In theservation that traces of fermentable carbo- experiments of Farkas and Fischer (121), suc-hydrate enhance the hydrogenlyase activity of cinate did not stimulate the partially inactivateddilute or aged cell suspensions (93, 112) may be system.accounted for on the basis of the latter function. Hoberman and Rittenberg believe that reac-

tivation of the exchange by organic compoundsC. The Exchange Reaction (and hydrosulfite) is due to reduction of inactive

Hydrogenase activity is usually defined as the "oxidized" hydrogenase to the active "reduced"enzymatic activation of molecular hydrogen enzyme and further suggest that the oxidation-either for participation in an exchange reaction reduction reactions occur in an iron porphyrinwith water or for reduction of an acceptor sub- prosthetic group. When cyanide was added tostance. There is little doubt that hydrogenase is suspensions of P. vulgaris under aerobic condi-also a necessary component of the enzyme com- tions (porphyrin iron presumably in the ferricplexes responsible for H2 production, and, conse- state), subsequent tests for exchange showedquently, the rate of H2 evolution may be a true complete lack of activity. Addition of cyanidemeasure of hydrogenase activity under certain to the actively exchanging system under an-conditions. aerobic conditions ("ferrous porphyrin"), on the

It seems to be generally assumed that the other hand, had no effect (120, 121). These re-ability to catalyze the exchange reaction between sults are similar to the observed effects of cyanideD2 and water (i.e., D2 + H20 = HD + HD0) on certain known iron porphyrin enzyme systems.is a fundamental property of hydrogenase and, Further evidence indicating an iron porphyrinaccordingly, that this reaction will always be prosthetic group was provided by the observationevident in any system containing the enzyme. that CO caused a strong inhibition which couldProbably owing to the relatively complex tech- be partially reversed by light (120). It should beniques involved, the exchange reaction has thus recalled in connection with this hypothesis thatfar been demonstrated and studied in only a few the nutritional experiments of Waring and Werk-bacterial species, primarily organisms of the man (107) showed that hydrogenase activitycolon-aerogenes group which are known to ac- with methylene blue as the acceptor was almosttivate H2 for reduction or to evolve H2 from entirely absent in iron deficient organisms.organic precursors.8 Joklik (122), however, has studied the effectsThe rate of the exchange reaction in resting of CO and cyanide on methylene blue reduction

suspensions of Proteus vukjaris is significantly by H2 in cell-free preparations of E. coli, anddepressed when the cells are preincubated in the his results are not consistent with the concept ofpresence of oxygen. This inactivation is re- an iron porphyrin prosthetic group. In the wellversible since the activity is restored by addition controlled experiments with the cell-free system,of hydrosulfite or by prolonged exposure to D2 cyanide inhibited aerated preparations onlyor H2 (120, 121). Hoberman and Rittenberg slightly and, in fact, appeared to be more ef-(120) reported that the exchange was also re- fective after prolonged anaerobic incubation.activated by glucose, pyruvate, formate, Moreover, the inhibition caused by CO was notfumarate, or succinate. It should be noted that reversed by light. These observations cast somethe organisms they used possessed hydrogenlyase doubt on the proposal of Hoberman and Ritten-activity; therefore, possibly with the first three berg and suggest that their results may be ex-compounds, dilution of deuterium in the gas plainable on a different basis, particularly sincephase was not entirely due to restoration of the objections based on considerations of O/R po-

8 The exchange reaction has also been demon- tentials have been raised against an iron por-strated in Rhodospirillum rubrum (119) and in phyrin prosthetic group.some of the organisms discussed in section II. 6. The exchange reaction between D2 and water


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probably represents the most sensitive indicator single unique mechanism exists for oxidation ofof hydrogenase activity and may be more valu- 12 with each particular oxidant. There are, ofable as such than tests for fixation of H', since course, numerous instances of analogous di-isotope discrimination may prove to be much versification in other phases of intermediarymore exaggerated using tritium as compared metabolism.with deuterium.' In this connection it is of inter- Since acceptors reduced as a result of hy-est that the experiments of Franke and M6nch drogenase activity are also reduced by oxidation(124) showed identical rates of utilization of D2 of organic compounds, it is reasonable to amendand H2 in the reduction of fumarate by intact the original schematic of Yamagata and Naka-cells of E. coli, i.e., no evident "isotope effect" mura by including the alternative pathway start-with these markedly different masses. Absence ing with a generalized donor, represented as XH2.of appreciable discrimination between deuterium It is evident that the first carrier reduced by oxi-and protium has also been recently reported for dation of XH2may or may not be identical withthe catalytic hydrogenation of quinone in non- that initially reduced by H2.biological systems (125). Previous attempts to elucidate the mechanism

of 12 oxidation have centered mainly on intactD. Oxidation of H2 with VGwuio Acceptor8 cell experiments with "specific" inhibitors (e.g.,The oxidation of H2, which is estimated by see 127, 128, 129). Undoubtedly, the terminal

manometric or other obvious types of measure- enzymes, and possibly also intermediary carriers,ments, is believed to be considerably more com- differ from acceptor to acceptor, and it mightplex than activation of H2 for exchange. It was therefore be expected that inhibitors would haveoriginally suggested by Yamagata and Naka- diverse effects depending on the nature of themura (126) that the reduction of acceptors by H2 ultimate oxidant. This is in fact observed to berequires several carriers and enzymes as indi- the case. Evaluation of the results of inhibitorcated by the (modified) schematic sequence experiments is further complicated by the fre-below. quently made observation that the effect of a

-+-- Intermediary carriers -+--~ Acceptor particular inhibitor with a given oxidant may* show a marked dependence on certain experi-T I mental factors, e.g., whether or not the cells are

Hydrogenase Specific preincubated in the presence of oxygen. Detailed(+ other enzymes?) . "acceptor enzymes" interpretation of the many ambiguous results

which have been reported will not be attemptedXH2 here; a number of other significant features of

This general mechanism presumes similarity in 12 oxidation by intact cells and cell-free systemsthe initial reactions, i.e., activation of H2 for re- are discussed below in connection with severalduction of a carrier, regardless of the nature of important acceptors.the terminal acceptor. After the initial activa- a. Fumarate. The rate of H2 oxidation withtion, the transport of electrons could occur fumarate by resting suspensions of P. vulgaris isthrough entirely different sequences, depending reported to be appreciably increased by additionon the acceptor and also on the particular type of fumarate to the growth medium (121). Sinceof organism under consideration. It has been the presence or absence of fumarate duringshown that the Knailgas reaction (2H2 + 02 - growth does not influence the level of exchange2120) in E. coli is completely inhibited by activity, it appears that in fumarate reductioncyanide, whereas the same reaction in BaciUu, the "semiadaptive" acceptor enzyme system isdelbrfickiiP is insensitive to this poison (126). generally rate-limiting. Nevertheless, the reduc-These observations indicate that with a given tion of fumarate competes with the exchangeterminal acceptor the same over-all reaction in reaction-the rate of exchange is usually lower indifferent organisms may involve different types the presence of fimarate, particularly in cellsof enzyme systems, and, accordingly, probably no which show high fumarate reduction activity.

9An example of isotope discrimination between Dissociable metal ions appear to be of im-deuterium and tritium in biochemical conversions portance in hydrogenase activity with fumarate.is found in the work of Verly et al. (123). Thus, partial inhibition of the reaction in crude


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extracts of E. coli is observed when complexing hydrogenase and methylene blue. Erlenmeyeragents such as dipyridyl are added (108). These et al. (133) suggest that there is an exchange ofcrude extracts contain suspended particles which, hydrogen between reduced (deuterated) methyl-as noted earlier, are the actual site of H, oxida- ene blue and water, and that the succinate be-tion with fumarate, nitrate, and ferricyanide comes "lighter" by virtue of reversibility of the(90). Lascelles and Still (129) have similarly reaction:found that fumarate reduction in intact cells andin extracts is inhibited by o-phenanthroline and succinate + MBa fumarate + MBH2also noted that addition of Fe++, Mn++, or Zn++ The experiments of Weinmann and co-workerscauses partial reversal; intact cell experiments (134) indicate that mammalian succinic dehydro-with oxygen and nitrate as acceptors showed genase may catalyze a "direct" exchange reactionessentially the same results. Since oxidation of H2 between succinate hydrogen and water since thewith methylene blue in cell-free preparations is process was found to occur in the absence ofunaffected by complexing agents (13), it seems dyes. However, crude preparations of the enzymelikely that inhibition of fumarate reduction by were used, and it is quite possible that an inter-such agents is due to combination with metal mediate natural carrier containing exchangeablecofactors associated with "acceptor enzymes". hydrogen participated in the reaction. The inter-Hydrogenase activity with fumarate in E. coli esting claim of Geib and Bonhoeffer (135) thatshows a disproportionate decline upon progres- succinic dehydrogenase preparations catalyze ansive dilution of the cell suspension; Lales and exchange between D, and water appears not toStill (129) have noted that the activity of diluted have been confirmed as yet.preparations is completely restored by addition of b. Nitrate. In the presence of H,, nitrate isboiled suspensions or by a combination of Mn " reduced by different strains of colon-aerogenesand diphosphopyridine nucleotide. The possible bacteria either to nitrite or to ammonia (128).role of pyridine nucleotides as carriers in hy- Organisms capable of producing the latter (viz.,drogenase activity will be discussed later. enteric bacteria and also certain clostridia) dis-The identity of the acceptor enzyme system in play the following reactions (136).

fumarate reduction is still unknown and couldeither be succinic dehydrogenase (acting in HNOs + 4H2 - NH, + 3H20reverse) or "fumaric hydrogenase" (fumaric HN02 + 3H2 NH,+ 2110reductase). The latter is a malonate insn tive+ H, - NH + Hflavin enzyme-thus far reported only in yeast- The last equation implies that hydroxylaminewhich transfers hydrogen from reduced dyes to may be a stage in the reduction of nitrate (orfumarate (130, 131).10 Attempts to differentiate nitrite) to ammonia, but attempts to demonstratebetween these two possibilities on the basis of intermediate formation of this compound havemalonate inhibition experiments have not beenii been unsuccessful (128, 136).successful (121, 126, 129). Many strains of colon-aerogenes organisms

Several studies on fumarate reduction with D, reduce nitrate in accord with the equation:in E. coli have disclosed that the succinate pro-duced contains much less deuterium than ex- HNOs + H, - HN0, +110pected on the basis of deuterium content ofthe gas phase (124, 132). This result indicates The stra ofE ical iu by Lacl and wasthat an intermediate is involved in which bound found to be incapable of oxidizing H awith nitritedeuterium is readily exchangeable with the light ou to be in e ofeoxrdiinwh t mtritehydrogen of the water solvent. Althoughsuccinic or hydroxylamine. However, in the presence of ahydrognoftewrssuitable redox dye (e.g., benzylviologen) nitrate,dehydrogenase has not been definitely implicated and hydroxylamine were reduced to

in fumarate reduction, it is pertinent to note that through hydrogenase action. This effect suggeststhe carbon-bound deuterium of deuterosuccinie that certanstrains are devoid ofintermiciaryacid becomes significantly diluted with ordinary certain stra red evoid ternediarehydrogen in the presence of succfinic de- carriers required for reduction beyond the itrite

10 The physiological reductant has not been It is presumed that nitrate reductase (nitratase)identified. is the acceptor enzyme in reduction of nitrate to


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nitrite by H2. Egami and Sato (137) have shown heavy bacterial suspensions but has no effect onthat reduced flavin adenine dinucleotide can act the rate of the exchange reaction (120).as hydrogen donor for nitrate reduction in cell- Extracts containing an active soluble hydro-free preparations of E. coli. Subsequent work by genase can be readily prepared from anaerobicallyEvans and Nason (138) with a purified nitrate grown E. coli (13, 142). In contrast to particulatereductase from Neurospora crassa similarly preparations from such cells, the soluble enzymedemonstrated that reduction of nitrate to nitrite reduces methylene blue rapidly but shows noin the presence of reduced triphosphopyridine activity with nitrate, fumarate, ferricyanide, ornucleotide is mediated by flavin adenine di- oxygen (90, 142). Cytochrome c, which is notnucleotide. Although no evidence is available, it characteristically present in E. coli, is also notis apparent that this may also be true in nitrate reduced (142). Reduction (reversible) of thereduction by H2. Inhibitor studies with cyanide, particulate cytochrome b1 through hydrogenaseaside, and CO as well as spectroscopic observa- action can, however, be observed in crude ex-tions indicate participation of iron porphyrin tracts and in intact cells by visual spectroscopicenzymes in nitrate reducing activity in colon- examination (13).aerogenes bacteria, and a close relationship be- Gest (13) and Joklik (142) were unable totween cytochrome b1 and nitrate reductase has demonstrate reduction of pyridine nucleotides bybeen postulated (139; see also 128). This view is H2 using cell-free preparations which showed highindirectly supported by the observation that activities with methylene blue as acceptor. Ahydrogenase activity with nitrate in E. coli is very low rate of H2 consumption, of doubtfulconfined to the particulate cytochrome b1-con- significance, was noted by Joklik upon addition oftaining fraction of the cells (90). The results of a diphosphopyridine nucleotide plus Straub's flavo-recent study of this relationship by Sato and protein to the hydrogenase preparation. From theNiwa (140) suggest the following sequence of preliminary experiments described by Vishniacelectron transport: Hydrogen donor-dehydro- and Ochoa (143), it appears that pyridinegenase . . . cytochrome b.. . nitrate reductase- nucleotides can be reduced by H2 oxidation innitrate. It is considered likely by these investi- cell-free systems containing bacterial preparationsgators that nitrate reductase itself is also an and enzyme supplements from other sources. Itiron enzyme. In this connection it should be noted should be noted that pyruvate and a number ofthat Granick and Gilder (141) have demonstrated other important metabolic intermediates whichthat iron protoporphyrin is essential either for can be reduced by specific pyridine nucleotideformation or activity of the nitrate reducing enzymes do not serve as acceptors for H2 eithermechanism in Hemophilus influenzae. The trans- in intact cells or unsupplemented cell-free prepa-port sequence noted above is of particular interest rations. This fact together with the other evi-in that it suggests that nitrate reductase may, dence available indicates to the reviewer thatunder certain conditions, replace "cytochrome hydrogenase is probably not a so-called "pyri-oxidase" as a terminal catalyst in facultative dine nucleotide enzyme". Although reduction ofanaerobes (139, 140). the nucleotides may be observed as an ultimate

c. Metlhylene blue and other acceptors. Hober- consequence of H2 oxidation under suitable cir-man and Rittenberg (120) observed that dilute cumstances, it is suggested that such reduction issuspensions of P. vulgaris, which could catalyze not obligatory in the normal activities of hydro-the exchange reaction, were incapable of reducing genase systems. The physiological significance ofmethylene blue at an appreciable rate. Methylene the observations reported by Vishniac and Ochoablue reduction did occur, however, in the presence remains to be established.of fumarate, and it was concluded that this com- The soluble hydrogenase of E. coli, strain B,pound could substitute for a natural carrier re. can be precipitated by ammonium sulfate, andquired for hydrogenation but not for exchange- the enzyme(s) has been partially purified (13,Further evidence for the relative complexity of 142). Previous attempts to demonstrate easilythe hydrogenation reaction is found in the ob- dissociable coenzymes (e.g., metal ions) with E.servation that urethane, at a proper concen- coli B preparations have given negative resultstration, inhibits H2 oxidation with the dye in (13), but recent studies indicate that essential co-


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factors may be more readily separated from the affect the activity of partially purified E. colienzyme in extracts from other strains of this hydrogenase and that the activity of the enzyme,organism (Peck, H. D., and Gest, H., unpub- inactivated to some extent by oxygenation, waslished data). not stimulated by cysteine and other "-SH re-As might be expected from the results of whole agents". Further work is obviously required to

cell experiments, the activity of the cell-free identify conclusively the functional groupshydrogenase of E. coli is adversely affected by affected by oxidizing agents.oxygen. The inactivation is at least partially Model systems have historically played anreversed by prolonged incubation with H2 (13, important role in the development of current142, 144). In Joklik's experiments (142), the ac- concepts of enzyme catalysis (33, 145), and it istivity of pre-aerated crude extracts was markedly to be hoped that information gained throughincreased by exposure to H2, an incubation study of such systems will aid in elucidation ofperiod of several hours (at 370?) being required the mechanism of biological activation of H2.for maximal effect. Incubation under H2 for only Prototypes of the three kinds of H2 "activation"five minutes in the presence of hydrosulfite considered in this review, viz., exchange, hydro-caused a comparable stimulation, whereas other genation, and evolution of H2, have been longreducing agents were ineffective under these con- known and studied in the field of heterogeneousditions. With the ammonium sulfate-precipitated (and homogeneous) metal catalysis'l; and theenzyme, on the other hand, reducing compounds obvious similarities between the biological andsuch as ferrous sulfate, thioglycolic acid and chemical processes have been recognised [e.g.,glutathione rapidly activated to the same degree see (145, 147, 148)]. The relative simplicity of theas prolonged exposure to H2. The stimulatory hydrogen molecule and of inorganic catalystsaction of thioglycolic acid was reported to be apparently led to the early belief that the activa-much greater under H2 than under N2, which tion of H2 on a metal surface must be a simpleindicates that the effect of "-SH reagents" is not reaction. Recent research, however, has showndue to simple direct reduction of functional that the processes involved are far more intricateenzymatic groups. than was originally envisioned and there are

Joklik (122, 142) has suggested that inactiva- many aspects of these catalyses which are stilltion of hydrogenase by oxygen can be adequately not well understood (149). In the biologicalexplained by oxidation of essential sulfhydryl activation (or production) of molecular hydrogen,groups on the enzyme. This view was supported the mechanism might be expected to be evenby the observation that various "-SE in- more complex if we consider the superimposedhibitors" (e.g., o-mercuribenzoate, o-iodoso- requirements of biochemical specificity.benzoate, phenylmercuric acetate) caused inhibi-tions of hydrogenase activity ranging from 30 to 6. HYDROGENASE IN ORGANISMS WHICH DO NOT85 per cent. Furthermore, it was reported that OXIDIZE NOR PRODUCE H2 DURINGthe inactivation by phenylmercuric acetate "NORMAL" METABOLIM(PMA) could be reversed by thioglycolic acid. The best example of this type is the non-In this experiment, the inhibition by PMA was symbiotic Nrfixing organism Azotbacer. Thisonly 28 per cent; addition of thioglycolic acidalone to the preparation increased the activity by *Thedecomposition of formats to 002 and H280 per cent, and the stimulated rate was not in the presence of rhodium catalysts was describedappreciably affected by further addition of PMA. by Blackadder in 1913 (146). References to earlierIt is evident that these complex results, as well as work on exchange and hydrogenation reactions inthose of the experiments summarized above, do homogeneous systems and a detailed analysis ofnot permit the unequivocal conclusion that the one such system can be found in the interestingoxygen-sensitivity can be entirely accounted for paper of Weller and Mills (125). The correspondingby oxidation of essential *ulfhydrl groups. E_ ]*reactions in heterogeneous systems were reviewedby oxidattiongessenthia sulfhydryn grfoups. Ei-t during a recent symposium dealing with surfacedence contradicting this suggestion is found in the reactions and are described in a series of papersexperiments of Gest (13), who observed that in the Journal of Physical and Colloid Chemistry,p-chloromercuribenzoate did not significantly 55, 1951.


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66 HOWARD GEST [voL. 18

strictly aerobic organism contains a powerful clarification of the still obscure nitrogenase-hydrogenase, particularly when grown under con- hydrogenase interrelationship.ditions where it must use N2 as the nitrogen The properties of the hydrogenase in cell-freesource. Cultivation in media containing am- preparations from Azotobaer, recently studied inmonium salts or other "fixed" nitrogen com- some detail (19, 151), appear to be generallypounds causes a marked depression in hydro- similar to those of the E. coli enzyme. In Azoto-genase content (150, 151, 152). This observation bacder, practically all of the enzyme is bound tosuggests a close relationship between H2 metabo- the insoluble particulate fraction. Particles fromlism and N2 fixation. Other types of evidence also A. vinelandii oxidize H2 in the presence of dyes,indicate such a relationship, viz.; (a) the corm- ferricyanide, cytochrome c (normally present inpetitive inhibition of N2 fixation by H2 in Azoto- the organism), and oxygen but not with nitrate,bacter and in symbiotic N2 fixation (153, 154), and fumarate, pyruvate, a-ketoglutarate, diphospho-(b) inhibition of light stimulated H2 production pyridine nucleotide or riboflavin. With ferricya-by N2 in photosynthetic bacteria (discussed in nide as the acceptor, a variety of potential co-section I. 1). factors including pyridine nucleotides, flavinThe early observations with Azotobacter sug- adenine dinucleotide, and riboflavin had no effect

gested that all organisms containing hydrogenase on the rate of H2 oxidation. As noted by Hynd-might also be capable of fixing N2. Recent tests of man et al. (19), failure of these compounds tothis hypothesis (155) clearly demonstrate that stimulate does not definitely exclude them ashydrogenase is present in organisms (e.g., E. coli) essential coenzymes since the presumably "or-which do not fix N2, at least according to present ganized" particles may already contain saturatingcriteria. In this connection, it is of interest that levels of firmly bound cofactors. Reduction (in"nitrogenaseless" mutants of Azotobacter, grown the absence of ferricyanide) of the nucleotideswith ammonia as the nitrogen source, contain and flavin compounds could not be detected byhydrogenase but in reduced quantity as com- spectrophotometric procedures. The observedpared with "wild type" or back mutants grown reduction of cytochrome c seems to be analogouswith N2 (150). It is evident that hydrogenase in to the already described cytochrome b1 reductionN2 fixing organisms must be of importance not in E. coli preparations. Spectroscopic studiesonly for the N2 reduction mechanism but also for (19) of "oxidized" A. vinelandii particles haveother reactions. disclosed a "hemoprotein" absorption peak atThere appear to be marked qualitative differ- 410 mni, which is shifted to 415 my upon incuba-

ences in the interaction of the Ns reduction and tion under H2. This spectral change could con-hydrogenase systems among the nitrogen fixing ceivably be associated with the hydrogenasebacteria. For example, the presence ofN2 prevents enzyme itself, but more likely it is due to indirectphotochemical H2 evolution by photosynthetic reduction of a distantly related porphyrinbacteria but does not affect H2 production from enzyme.pyruvate by C. pasteurianum (29). Another Oxidation of H2 with ferricyanide by thestriking apparent contradiction is found in the particles from A. vinelandii is noncompetitivelyeffects of H2 on N2 fixation; there is clear-cut inhibited by CO, and the inhibition is not reversedcompetitive inhibition in Azotobacer and in by light (19). Similar results with intact cellssymbiotic systems, but none with the anaerobic using 02 as the acceptor were described by Wilsonclostridia and photosynthetic bacteria (66, 156), and Wilson (157). Green and Wilson (151) foundpossibly because of complications arising from H2 that certain cell-free preparations from A. agile,evolution by these organisms (156). At present which could not oxidize H2 with methylene blue,there is no unambiguous explanation of these were activated by incubation under H2 or, in somedifferences, but it seems possible that they are instances, by addition of cysteine. The possibilityprimarily of a quantitative nature rather than that sulfhydryl groups are of some importance inqualitative. The influence of the nitrogen source the activity of Aotobater hydrogenase is indi-for growth on hydrogenase content of clostridia cated by the fact that p-chloromercuribenzoate,and photosynthetic bacteria has not yet been at a proper concentration, strongly inhibitssystematically investigated; it is likely that in- methylene blue reduction by A. vinelandii prepa-formation on this point will be of some value in rations. This inhibition, which is reversed by


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cysteine, is strkingly evident only when the by the flagellate green alga, Chlamydo-"-SH inhibitor" is preincubated with the monas Moewuaii. Arch. Biochem. Bio-hydrogenase preparation in the presence of phys., 38, 219-230.oxygen; addition of the inhibitor to the system 4. KAMEN, M. D., AND GEST, H. 1952 Seren-under an atmosphere of .2 caused only a 16 dipic aspects of recent nutritional researchpercentdeprssioofctivty(in bacterial photosynthesis. In Phos-

per cent depression of actaivty (151). phorus metaboli8m, pp. 507-519. Vol. II.Contrary to expectations, particulateprepra Edited by W. D. McElroy and B. Glass.

rations from A. vinekzndii, which were very active The Johns Hopkins press, Baltimore, Md.with regard to H2 oxidation with acceptors, 5. NAKAMURA, H. 1939 Weitere Untersuch-catalyzed the exch reaction at a very slow ungen uber den Wasserstoffumsatz bei denrate (19). The significance of this observation is Purpurbakterien, nebst einer Bemerkungnot evident, and the authors (19) could suggest fiber die gegenseitige Beziehung zwischenonly that the low rate observed may have bsen Thio-und Athiorhodaceen. Acta Phyto-due to nonoptimal experimental conditions. chim. (Japan), 11, 109-125.Hydrogenase activity (i.e., exchange, reduc- 6. NAKAMURA, H. 1941 Weitere Untersuch-

tion, or fixation ofH.)has also ben noted i ungen uiber die bakterielle Photosynthese., orhfichioneift. her, also beenortpossess Acta Phytochim. (Japan), 12, 43-64.organisms which neither fix N2 nor posses a 7. GEST, H. 1951 Metabolic patterns in"normal" H2 metabolism, specifically in Aceto- photosynthetic bacteria. Bacteriol. Revs.,bader peroxydams (158), Bacillus acidi ldici' 15,183-210.(120, 159, 160), Bacillus delbrckiis (126), S. 8. BREGOFF, H. M., AND KAMEN, M. D. 1952faecalis (161), and certain luminous bacteria Photohydrogen production in Chromatium.(162). In addition, Boichenko (163, 164, 165) has J. Bacteriol., 63, 147-149.reported that chloroplast preparations from 9. GnsT, H., AND KAMEN, M. D. 1949 Photo-higher plants display hydrogenate activity; con- chemical production of molecular hydrogen. . sss r E * ~~~by growing cultures of photosyntheticfirmation of this observation would be of obvious bacteria. J. Bacteriol., 58, 39-245.importance to the comparative biochemistry of 10. BREGOFF, H. M., AND KAMEN, M. D. 195212 metabolism. Quantitative relations between malateThe fact that H2 has not been encountered as dissimilation, photoproduction of hydro-

an intermediate or end product in what is con- gen, and nitrogen metabolism in Rhodo-sidered to be the normal metabolism of these spirillum rubrum. Arch. Biochem. Bio-organisms suggests that hydrogenase activity in phys., 36, 202-220.these instances may reflect lack of specificity of 11. NAKAnuRA, H. 1938 tJber die Rolle deran enzyme which ordinarily is concerned with Hydrogenase im Stoffwechsel von Rhodo-other substrates. Although there is no direct evi- bacillus palustris. Acta Phytochim.dence on this question, it seems worthwhile to (Japan), 10, 259-270.

.,.,iderthispossibility. 12. GEST, H. 1950 Anaerobic oxidation ofmalate and hydrogen in the dark by

ACKNOWLEDGMENT RhodospiriUum rubrum. Bacteriol. Proc.,1950, 136-137.

The author is indebted to his colleagues, Drs. 13. GEsT, H. 1952 Properties of cell-freeS. S. Barkuii, J. L. Karison, L. 0. Krmpitz hydrogenases of Escherichia coli andH. D. Peck, and H. E. Swim, for stimulatg RhodopiriUum rubrum. J. Bacteriol., 63,discussions which were very helpful in the devel- 111-121.opment of this review. 14. GEST, H. 1951 Enzymatic oxidation of

molecular hydrogen by bacterial extracts.REFERENCES Federation Proc., 10, 188.

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Gattung Hydrogenomonas agilis. Centr. Cl. acetobutylicum. 3. Potassium as anf. Bakt., Abt. II, 40, 430-433. essential factor in the fermentation of

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43. HARDEN, A. 1930 The metabolism of bac- reaction to several bacterial species.teria. In A system of bacteriology in rela- Arch. Biochem., 16, 473-474.tion to medicine. Vol. L Refer to Chap- 59. SoumRs, T. L., AND TRANO, Y. 1953ter VI. His Majesty's Stationery Office, Reduction of methionine sulfoxides byLondon, England. Escherichia coli. Arch. Biochem. Bio-

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45. BARKuLus, S. S., AND GEST, H. 1953 The 61. FOuiBERT, E. L., JR., AND DOUGLAS, H. C.role of C2-derivatives in the decomposition 1948 Studies on the anaerobic micrococci.of formate. Biochim. et Biophys. Acta, I. Taxonomic considerations. J. Bac-11 306-3. teriol., 56, 25-34.

46. UTTER, M. F., AND WOOD, H. G. 1951 62. JOHNS, A. T. 1951 The mechanism ofMechanisms of fixation of carbon dioxide propionic acid formation by Veillonellaby heterotrophs and autotrophs. Ad- gazogenes. J. Gen. Microbiol., 5, 326-336.vances in Enzymol., 12, 41-151. 63. TABACHNICK, J., AND VAUGHN, R. H. 1948

47. WIERINGA, K. T. 1940 The formation of Characteristics of tartrate-fermenting spe-acetic acid from carbon dioxide and hydro- cies of Clostridium. J. Bacteriol., 56,gen by anaerobic spore-forming bacteria. 435-443.Antonie van Leeuwenhoek, J. Microbiol. 64. KOHLLER, E. F., JR., AND GEST, H. 1951Serol., 6, 251-262. A comparative study of the light and dark

48. KARLssoN,J. L., VOLCANI, B. E.,AND BARKER, fermentations of organic acids by Rhodo-H. A. 1948 The nutritional requirements spirillum rubrum. J. Bacteriol., 61, 269-of Clostridium aceticum. J. Bacteriol., 56, 282.781-782. 65. GEST, H., AND KAMEN, M. D. 1949 Photo-

49. BARKER, H. A. 1937 On the fermentation production of molecular hydrogen byof glutamic acid. Enzymologia, 2, 175-182. Rhodospirillum rubrum. Science, 109, 558-

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57. HOOGERHEIDE, J. C., AND KocHoIATY, W. 72. BARKER, H. A. 1943 The influence of car-1938 Reduction of amino-acids with gase- bon dioxide concentration on the rate ofous hydrogen by suspensions of Cl. sporo- carbon dioxide reduction by moleculargenes. Biochem. J., 32, 949-957. hydrogen. Proc. Natl. Acad. Sci. U. S.,

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kingen over de Methaangisting. Disserta- composition of formic acid. Biochem.tion, Delft. J., 23, 1187-1198.

74. KLUYVER, A. J., AND SCHNELLEN, C. G. T. P. 89. KALNITSKY, G., AND WERKMAN, C. H. 19431947 On the fermentation of carbon mon- The anaerobic dissimilation of pyruvateoxide by pure cultures of methane bacteria. by a cell-free extract of Escherichia coli.Arch. Biochem., 14, 57-70. Arch. Biochem., 2, 113-124.

75. STADTMAN, T. C., AND BARKER, H. A. 1951 90. GEST, H. 1952 Molecular hydrogen: oxida-A new formate-decomposing bacterium, tion and formation in cell-free systems.Methanococcus vannielii. J. Bacteriol., Phosphorus metabolism, pp. 522-543. Vol.62, 269-280. II. Edited by W. D. McElroy and B.

76. STEPHENSON, M., AND STICKLAND, L. H. Glass. The Johns Hopkins Press, Balti-1933 Hydrogenase. III. The bacterial more, Md.formation of methane by the reduction of 91. YUDKIN, J. 1932 Hydrogenlyases. II. Someone-carbon compounds by molecular hy- factors concerned in the production ofdrogen. Biochem. J., 27, 1517-1527. the enzymes. Biochem. J., 26, 1859-1871.

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78. DAVISON, D. C. 1951 Studies on plant 151-161.formic dehydrogenase. Biochem. J., 49, 93. LASCELLES, J. 1948 Studies on formic520-526. hydrogenlyase in washed suspensions of

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80. PAEx:S, W. C. C., AND JOLLYMAN, W. H. PITZ, L. 0. 1950 Fixation of formic acid1901 The bacterial decomposition of in pyruvate by a reaction not utilizingformic acid into carbon dioxide and hydro- acetyl phosphate. J. Biol. Chem., 182,gen. J. Chem. Soc., 79, 386-391. 525-540.

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82. GORDON, J., AND STICKLAND, L. H. 1949 chem. J., 27, 1528-1532.Glycine and formic hydrogenlyase. J. 96. PINSKY, M. J., AND STOKES, J. L. 1952Hyg., 47, 278-280. The influence of age on enzymatic adapta-

83. STEPHENSON, M., AND STICKLAND, L. H. tion in microorganisms. J. Bacteriol.,1932 Hydrogenlyases. Bacterial enzymes 64, 337345.liberating molecular hydrogen. Biochem. 97. LICHSTEIN, H. C., AND BOYD, R. B. 1953J., 26, 712-724. The formic hydrogenlyase system of

84. STEPHENSON, M., AND STICKLAND, L. H. Aerobacter aerogenes. J. Bacteriol., 65,1931 Hydrogenase: a bacterial enzyme 617-618.activating molecular hydrogen. I. The 98. BILLEN, D., AND LICHSTEIN, H. C. 1951properties of the enzyme. Biochem. J., -Nutritional requirements for the produc-25, 205-214. tion of formic hydrogenlyase, formic de-

85. STEPHENSON, M. 1937 Formic hydrogenly- hydrogenase, and hydrogenase in Escher-ase. Ergeb. Enzymforsch., 6, 139-156. ichia coli. J. Bacteriol., 61, 515-522.

86. ORDAL, E. J., AND HALVORSON, H. 0. 1939 99. KuSHNER, D. J., AND QUASTEL, J. H. 1953A comparison of hydrogen production from Factors underlying bacterial enzyme syn-sugarsandfomthesis. Proc. Soc. Exptl. Biol. Med., 82,sugars and formic acid by normal and 388-392.

variant strains ofE3c8erichia coli.9. 100. GRUNBERO-MANAGO, M., SZULMAJSTER, J.,Bacteriol., 38, 199-220. AND PROUVOST, A. 1951 Hydrogenlyase,

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88. STICKLAND, L. H. 1929 The bacterial de- Nutritional requirements for hydrogenase


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tfber das dehydrierende Enzymsystem Leuchtbakterien. I. Ann. Chem., 635,von Acetobacter peroxydams. 1. tVber den 122-149.Mechanismus der Oxydationsvorghnge. 163. BoICHENKO, E. A. 1946 Evolution ofXLIV. Ann. Chem., 522,116-137. hydrogen by isolated chloroplasts.

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162. CLAREN, 0. B. 1938 Zum Stoffwechsel der 5452 g (1949). Seen in abstract only.


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