Proc. Natl. Acad. Sci. USAVol. 88, pp. 6122-6126, July 1991Biochemistry

The heme groups of cytochrome o from Escherichia coli(cytochrome oxidase/quinol oxidase/heme A/heme 0)

ANNE PUUSTINEN AND MARTEN WIKSTROMHelsinki Bioenergetics Group, Department of Medical Chemistry, University of Helsinki, Siltavuorenpenger 10A, SF-00170 Helsinki, Finland

Communicated by Britton Chance, April 4, 1991 (receivedfor review January 16, 1991)

ABSTRACT Cytochrome o, one of the two terminalubiquinol oxidases of Escherichia cofi, is structurally andfunctionally related to cytochrome c oxidase of mitochondriaand some bacteria. It has two heme groups, one of which bindsCO and forms a binuclear oxygen reaction center with copper.The other heme is unreactive toward ligands, exhibits stronginteractions with the binuclear center, and is mainly respon-sible for the reduced-minus-oxidized a band. Protoheme hasbeen thought to be the prosthetic group of b-type cytochromes,including cytochrome o. However, the hemes of cytochrome oare of a different kind, for which we propose the name heme0. Its pyridine hemochrome spectrum is blue-shifted by 4 nmrelative to that of protoheme, and chromatographic behaviorshowed that it is much more hydrophobic than protoheme. Fastatom bombardment mass spectrometry yielded a molecularmass of 839 Da. Heme 0 is proposed to be a heme A-likemolecule, containing a 17-carbon hydroxyethylfarnesyl sidechain, but with a methyl residue replacing the formyl group.

Escherichia coli contains two terminal oxidases, both ofwhich oxidize ubiquinol by molecular oxygen. One of themwas called cytochrome o (for oxidase) by Castor and Chance(1), who described its CO-binding properties. The genes forcytochrome o have been elucidated (2), and strong protein-structural homology has been found with cytochrome coxidase (cytochrome aa3) of mitochondria and some bacteria(2, 3), especially for the heme-binding largest subunit. Theother quinol oxidase of E. coli, cytochrome d (or bd), isstructurally and functionally unrelated to cytochrome aa3(4-6). Cytochrome o contains two hemes. One binds CO (1)and probably forms a binuclear 02 reaction center togetherwith a copper ion (7). The other heme is probably a low-spinsix-coordinated hemochrome, as revealed both by its EPRand optical spectra (6-8). Cytochrome o has only one copperper two hemes and lacks the CUA center typical of cy-tochrome aa3 (6, 9). It functions as a proton pump much likecytochrome aa3 (6, 10).Thus cytochrome o is both structurally and functionally

homologous to cytochrome aa3, but it exhibits distinct dif-ferences as well. We have undertaken studies of cytochromeo to gain more insight into the structure and function ofterminal proton-translocating oxidases, by their comparison.In this paper we report on the spectral, redox, and structuralproperties of the heme groups.The spectral properties of cytochrome o have been con-

troversial. The reduced-minus-oxidized a band is split at 77K into peaks at -555 and 561 nm. The 555-nm species hasbeen ascribed to the CO-reactive high-spin heme (11, 12),which according to Salerno et al. (12) has a midpoint redoxpotential relative to the normal hydrogen electrode (Em) atpH 7 of -160 mV. On the other hand, Withers and Bragg (13)attributed the long wavelength species to the oxygen-reactiveheme, to which they ascribed a CO-sensitive midpoint po-

tential (265 mV). The other heme species exhibited an Em of140 mV. On the basis of the potentiometric behavior of thehigh-spin signal of the oxygen-reacting heme, Salerno et al.(7, 12) concluded that the Em for Cu in the binuclear site is=350 mV. They further demonstrated that the enzyme ex-hibits anticooperative heme-heme and high-spin heme-copper interactions, similar to those in cytochrome aa3.We recently suggested that the entire reduced-minus-

oxidized a band, including its 555- and 561-nm components,is due almost entirely to the six-coordinated low-spin heme,whereas both hemes contribute about equally to the Soretband (6). However, the a band ofthe reduced-minus-oxidizedenzyme behaves inhomogeneously, both spectrally and po-tentiometrically (12, 13). With Salerno et al. (7, 12), weascribe this to combined spectral and redox potential inter-actions between the low-spin heme and the binuclear center.CO has been described to have small and somewhat

variable effects on the spectrum of the reduced enzyme in thea band (11, 14-17). Here we study the CO-difference spec-trum in some detail and report its relevant specific absorp-tivities.The hemes of cytochrome o have been generally thought to

be protohemes (see e.g., refs. 18 and 19). However, scruti-nization of the cited data indicates that an actual determina-tion of the heme type has not been made previously* and thatthe anomalously blue-shifted pyridine hemochrome (6) hasbeen unnoticed or neglected. Here we propose a structure forthe prosthetic heme groups of cytochrome o.

MATERIALS AND METHODSDeoxycholate-washed (20) membranes from E. coli strain RG145 (cyd-) (ref. 21; for growth conditions see ref. 6) werefurther purified with cold acetone/NH40H, and the hemeswere extracted from the pellet into cold acetone/HCI asdescribed by Weinstein and Beale (22). Alternatively, prep-arations ofpurified cytochrome o (6), sperm whale myoglobin(Sigma), or bovine heart cytochrome oxidase (23) were used.The hemes in acetone/HCI were extracted into ether, and theheme-containing upper phase was washed with water (22).The ether was evaporated under a stream of nitrogen. Thehemes were dissolved in ethanol/methylene chloride (50:50,vol/vol) and applied to a 1.5-ml bed volume column ofDEAE-Sepharose CL-6B (acetate) (24), which had beenequilibrated with ethanol/methylene chloride (25). The col-umn was washed with 20 ml of the equilibration solution,followed by 3 ml of aqueous ethanol, and the hemes wereeluted in ethanol/acetic acid/water (70:17:13, vol/vol) (25).The hemes were separated from each other by reverse-

phase HPLC by using an Altex Ultrasphere ODS column (25cm x 4.6 mm) with 5-,um particles. The solvent was 95%

Abbreviations: Em, midpoint redox potential relative to the normalhydrogen electrode; TMPD, tetramethyl-p-phenylenediamine.*In the course of this work we noted that Castor and Chance (1),referring to unpublished work of L. Smith, reported that the hemesof "cytochrome o" are not protohemes. To our knowledge this hasbeen overlooked and not followed up subsequently.

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A

AA =0.02f

Il

I

.1

400 440 480 520Wavelength, nm

B

ethanol/acetic acid/water (70:17:7, vol/vol), and the flowrate was 0.6 ml/min. Heme fractions were detected byabsorbance at 402 nm, collected, and evaporated in a rotaryevaporator.

ll+ Fast atom bombardment mass spectra were recorded at theAA= State Technical Research Center (VTT; Espoo, Finland) by0.002 using a JEOL SX 102 mass spectrometer. HPLC-purified

protoheme (from myoglobin) and heme 0 (see above) weredissolved into a glycerol matrix on the sample plate. Thesample was bombarded by Xe atoms at an accelerationvoltage of 10 kV and a fast atom bombardment gun voltageof 3 kV, using the positive ion mode. The temperature of theion source was 530C, and the scan range was 20-2000 m/z.The data was processed with a Hewlett-Packard 9000 com-....... . ,puter.

Optical spectra were recorded using a Shimadzu UV-3000instrument. Dual wavelength spectrophotometry was carriedout by using a DBS-1 (Johnson Foundation Workshop,University of Pennsylvania) spectrophotometer. All opticalspectroscopy was performed at room temperature in cuvetteswith a 1-cm light path length.

CO-difference spectra were obtained by first recording thebaseline of the anaerobically reduced sample in the Shimadzu(Kyoto) instrument at a 0.5-nm slit width [tetramethyl-p-

560 600 phenylene diamine (TMPD) plus ascorbate, see the legend toFig. 1], by subsequent slow bubbling of CO gas through thesample for 6 min in the dark, and by finally recording thedifference spectrum. A second spectrum, routinely recordedafter a further CO treatment for 6 min, showed no difference

T from the first.AA = Pyridine hemochrome spectra were recorded as described0.002 by Berry and Trumpower (26).

A 1

AI RESULTS AND DISCUSSION

480 520Wavelength, nm

C

AA =

0.001556-565 nm

f

1 minP.,

FIG. 1. Reduced-minus-oxidized and CO-diffisolated cytochrome o. The medium contained 40 n(wt/vol) dodecyl maltoside at pH 7.4, and 0.44 ,uMThe baseline was recorded .... ), followed by aascorbate and 0.4 mM TMPD. After anaerobiominus-oxidized difference spectrum was recordedspectrum was memorized as a new baseline, aktreated with CO (see Materials and Methods), an(minus reduced spectrum was recorded (----). (Iwere identical to those in A, except for the concentwhich was 0.33 ,uM. The anaerobically reducedspectrum was recorded as before (----) and mem(afew grains of solid sodium dithionite were added, areduced minus anaerobically reduced differenc

U1>, Spectral Properties of the Low- and High-Spin Hemes of" __ Cytochrome o. Fig. 1A shows the reduced-minus-oxidizedT\AA_ difference spectrum of the purified cytochrome o preparation

AA = (6). It is important to note that the enzyme was reduced0.001 anaerobically with TMPD plus ascorbate (see below). The a

band is broad at room temperature; it is known from previouswork that two peaks can be distinguished at 77 K (11, 12, 17).The Soret/a-band ratio is 11.8, in the same range as found by

50

, k others (10.6-12.2; cf. refs. 1, 11, and 15), but much higher560 600 than for cytochrome aa3 (27). The specific absorptivity of the

a band (reduced minus oxidized; 560 minus 580 nm) is about24 mM-1 cm-1 on the basis of concentration of enzyme (allspecific absorptivities reported here are based on the con-centration of enzyme, containing two hemes), which was inturn deduced from pyridine hemochrome determination (6).The corresponding value for the Soret band (reduced minusoxidized; 427 minus 460 nm) is then 283 mM-1 cm-1. This issimilar to values calculated from spectra reported in refs. 11

[02N 0 and 15.The CO-difference spectrum (Fig. 1A) of the reduced

cytochrome o is very similar to the photochemical actionspectrum measured by Castor and Chance (1). It is important

erence spectra of to note that the red region of the spectrum does not reveal anynM Tris Cl, 0.05% distinct trough below the baseline, but it shows discrete a andcytochrome o. (A) P3 bands at 568 and 535 nm, respectively. This supports theaddition of 2 mM previous contention that the CO-reactive high-spin hemesis, the reduced- contributes little, if any, to the reduced-minus-oxidized dif-i- . nen tnisd the sample wasd the CO-reduced9) The conditions.ration of enzyme,(minus oxidized)orized. After this,and the dithionite-e spectrum was

recorded ( ). (C) The time course of the aerobic-anaerobictransition was followed at 556-565 nm. The medium was as describedabove; TMPD, ascorbate, and enzyme concentrations were 0.4 mM,10 mM, and 0.8 uM, respectively. Points of anaerobiosis are indi-cated; at +02, dioxygen was added by stirring. The absorbanceincreases at 556 and 565 nm correspond to a downward and upwarddeflection, respectively.

TAA =0.01I

400 440

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6124 Biochemistry: Puustinen and Wikstrom

ference spectrum in the 500- to 600-nm region (6). Its con-tribution to the Soret band is near 50% (Fig. PA).The a and (3 peaks of the CO-difference spectrum both

have specific absorptivities of about one-half of the reduced-minus-oxidized 560-nm band. Here we differ from both Kitaet al. (11) and Matsushita et al. (15), who found substantiallylower specific absorptivities. We find the peak-to-troughabsorptivity of the Soret CO-difference spectrum to be about287 mM-1cm-1. This is again much higher than valuesreported previously (11, 15, 28). However, the ratio of thepeak-to-trough in the Soret band to the extent of the a peakis 23.7 (Fig. P1), which is similar to earlier data (11, 15, 28).Therefore, we ascribe the higher molar absorptivities foundhere to more complete occupancy of the CO adduct.A Contaminating, Low-Potential Cytochrome b-556. In con-

trast to the experiment above, reduction of cytochrome o forspectroscopic analysis has usually been routinely performedwith dithionite. Addition of dithionite to the reduced anaer-obic sample caused further reduction of a cytochrome bcomponent with a sharp a-peak maximum at 556 nm and aSoret maximum at 424 nm in the difference spectrum (Fig.1B). This species is also hardly reduced anaerobically withubiquinol 1 and does not react with CO. Apparently it has anEm significantly lower than that of the other species. Itsabsorbance varies between 20o and 30%o of the total dithio-nite-reduced minus oxidized a band in our enzyme prepara-tions. It is thus clearly substoichiometric with respect to thecytochrome o hemes (see also below). The cytochrome b-556has probably contributed to spectral and potentiometricmultiplicity of b cytochromes in at least some cytochrome opreparations reported previously and to variations in suchmultiplicity between different enzyme preparations (see e.g.,ref. 13). We ascribe cytochrome b-556 to a low-potentialb-type cytochrome that may contaminate most cytochrome opreparations. It might yet be of functional importance forquinol oxidase activity (see below). Kita et al. (29) havepurified and characterized a b-type cytochrome (which theycalled b-556) from E. coli, which has spectral and redoxproperties very similar to those of cytochrome b-556 de-scribed here.The Basis for the Asymmetric Behavior of the 560-nm Band.

Fig. 1C shows that, upon anaerobiosis with TMPD plusascorbate, the 560-nm band (Fig. 1 A and B) is formedasymmetrically. This was determined by the dual-wavelengthtechnique with the two wavelengths symmetrically displacedfrom the center of the absorption band. The absorbanceincrease at anaerobiosis starts at the longer wavelengths ofthis band (initial upward deflection at 02 = 0), followed by aslower (later) absorbance increase at the shorter wavelengths(secondary downward deflection). These kinetics do notinvolve the b-556 component, which is insignificantly re-duced in these conditions and on this time scale. This resultsuggests a spectral contribution to the 560-nm band from atleast two heme components: a long-wavelength species withhigh Em and a short-wavelength species with lower Em.Either both hemes of the enzyme contribute significantly tothe 560-nm band, or then there must be a combination ofspectral and redox interactions between the low-spin hemeand the binuclear oxygen reaction site. Since we have ex-cluded the former possibility (above), the latter must be thecase.

Previously, the 02- and CO-binding heme has been vari-ably assigned to the long- and short-wavelength componentsof the 560-nm band, respectively (see Introduction), based onthe spectrally and potentiometrically heterogeneous behav-ior of the band, also reported here, its splitting at 77 K, andthe CO-difference spectrum. On the basis of our data, weascribe the heterogeneity exclusively to center-center inter-actions in the enzyme (see also ref. 7). Thus both the Em andthe position of the a band of the low-spin heme depend on

whether the heme (and/or copper) of the binuclear site isoxidized or reduced. In the former case, the Em is high andthe a-absorption peak(s) are red-shifted; in the latter case, theopposite is true. The splitting of the 560-nm band at 77 K isthen probably due to an asymmetrical optical transition, alsoexhibited by the low-potential b-type heme ofthe cytochromebc, complex (30).Cytochrome o Contains Heme 0. The hemes of cytochrome

o have been thought to be protohemes as is thought togenerally be the case for the hemes of b-type cytochromes(see e.g., ref. 31). Hemes extracted from cytochrome o andfrom membranes ofE. coli (see Materials and Methods) wererun on an HPLC column that readily separates protohemefrom heme A. As seen in Fig. 2, our enzyme preparationcontains two kinds of heme, one of which runs identicallywith protoheme, but it accounts for only -25-30%o of thetotal. The main heme fraction (heme 0, see below) is highlyhydrophobic; it is retained on the column even longer thanheme A. The amount of protoheme coincides with theoptically determined amount of low-potential cytochromeb-556 species described above. In preliminary work, cy-tochrome b-556 has been separated from the cytochrome oentity; pyridine hemochrome determinations have confirmedthat b-556 indeed contains protoheme (A.P., unpublishedresults).

A

B

-I10 20 30 40 50 60 70 80

Time, min

FIG. 2. Reverse-phase HPLC elution profiles of hemes. Thehemes were extracted from purified cytochrome o (A) and frommyoglobin and cytochrome c oxidase, the hemes of which werecombined (B). Elution times for protoheme, heme A, and heme 0were 29, 61, and 71 min, respectively. In A, the integrated areas ofthe protoheme and heme 0 peaks were 27% and 63%, respectively,of the total integrated heme absorbance at 402 nm.

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Proc. Natl. Acad. Sci. USA 88 (1991) 6125

fIIII

II

T

A = 0.005

510 530 550 570Wavelength, nm

590

FIG. 3. Dithionite-reduced minus oxidized pyridine hemochromespectra of heme 0 ( ) and protoheme (----) from HPLC-purifiedheme fractions.

Fig. 3 shows that the pyridine hemochrome spectrum ofpurified heme 0 is blue-shifted by 4 nm relative to proto-heme. The hemes of cytochrome o must have a structuredifferent from protoheme, which we tentatively call heme 0.tA Possible Structure of Heme 0. Pyridine hemochrome

spectra are very sensitive to the nature and number ofelectron-attracting groups on the porphyrin ring, and thisphenomenon has been extensively studied in the past (see ref.34). For example, we can readily exclude the presence of a

formyl group, which due to its very strong electron-attractingpower displaces pyridine hemochrome and native spectra farto the red, as in heme A. The pyridine hemochrome spectrumof protoheme is much less red-shifted, but the peak positionis yet further to the red than for many other hemes. This isdue to the electron-attracting vinyl groups in the 2 and 4positions of the porphyrin ring (34). The pyridine hemo-chrome spectrum of heme 0 is blue-shifted by 4 nm from this(Fig. 3) and is very similar to that of 2(4)-hydroxyethyl-4(2)-vinyl deuteroheme. However, the blue shift is smaller than in

tAccording to current convention, both isolated heme structures andcytochromes are named a, b, c, and d (31). However, in agreementwith Caughey et al. (32), we prefer the use of capital letters todescribe the heme structure as isolated (33). Lowercase letters maythen be freely used for cytochromes and enzymes, as well as todescribe individual protein-bound heme groups (for example, cy-

tochrome bc, and aa3 complexes, cytochrome b5, heme cl of the bc,complex, heme a3 of the aa3 complex, etc). On the basis of thestrong homology between cytochrome o and cytochrome aa3-typeoxidases and the distinct heme 0 structure of the former, we

tentatively suggest that cytochrome o be termed cytochrome 003,

which also appears historically pertinent. Thus heme o is thelow-spin hemochrome-type species that does not react with ligandsand heme 03 is the high-spin CO- and 02-reactive heme of thebinuclear site, both having the heme 0 structure as isolated.

hematoheme, where positions 2 and 4 are both occupied byhydroxyethyl groups (34). This comparison suggests that onebut not both vinyl groups of protoheme has been replaced inheme 0 by a less electron-attracting residue, possibly a(substituted) hydroxyethyl group.Protoheme is one of the most hydrophobic naturally oc-

curring hemes. In this respect, it is surpassed only by hemeA, the hydrophobicity of which is mainly due to its longhydroxyalkyl side chain. The behavior of heme 0 on HPLC(Fig. 2) therefore strongly suggests the presence of a similarside chain. Combining this with the information from thepyridine hemochrome spectrum allows the conjecture thatheme 0 might differ from protoheme by having one vinylgroup of the latter replaced by a long hydroxyalkyl side chain.A minimum hypothesis would further suggest that heme 0may have a methyl group in position 8, as in protoheme, inplace of the formyl group of heme A.A long hydroxyalkyl side chain would increase the molec-

ular mass of heme 0 much above that of protoheme. Fastatom bombardment mass spectrometry of isolated andHPLC-purified protoheme from myoglobin yielded a mass of617 Da for the molecular ion, which is the expected value.The corresponding mass of purified heme 0 was 839 Da,which strongly suggests the presence of a long alkyl sidechain. The molecular mass of heme 0 is 14 Da smaller thanthat of heme A. This is consistent with a heme 0 structurethat differs from heme A only by having the formyl group ofthe latter replaced by a methyl group. Thus heme 0 isproposed to have a hydroxyethylfamesyl side chain identicalto that in heme A in one of the positions (2 or 4) occupied bya vinyl group in protoheme. The analogy to heme A makessubstitution at position 2 more likely. A methyl group inposition 8, instead of formyl, could explain why heme 0appears more hydrophobic than heme A. We conclude thatthe proposed structure is consistent with all the availabledata, but that it still needs to be proven unambiguously byNMR spectroscopy.

Conclusions. We conclude that the cytochrome o entitycontains two hemes of the 0 type. Our enzyme preparationfurther contains variable, substoichiometric amounts of alow-potential cytochrome b-556 species, the prosthetic groupof which is protoheme. This species is also present in at leastsome ofthe cytochrome o preparations described earlier (13).We have found that the b-556 can be separated from thecytochrome o entity; this appears to be associated with adecrease of ubiquinol oxidase activity (A.P., unpublishedresults). Cytochrome b-556 might be the entity that oxidizesquinol and delivers the electrons to the cytochrome o com-plex.The two heme 0 groups have different properties when

bound to the enzyme (cf. hemes A in cytochrome aa3). Weconclude that the low-spin heme is the main contributor to thea band of reduced-minus-oxidized enzyme with very little, ifany, contribution from the high-spin CO- and 02-bindingheme. In the Soret, both hemes contribute nearly equally tothe reduced-minus-oxidized band. Yet, the a band behavesheterogeneously both spectrally and as a redox system. Weascribe this to interactions between the low-spin heme andthe binuclear heme-copper center (cf. refs. 7 and 12). Allthese properties are also typical for the cytochrome aa3system (33).The discovery of heme 0 in cytochrome o provides yet

another similarity to cytochrome aa3-type oxidases. Thesenow comprise primary protein structure, especially subunit I(2, 3), function (proton pumping; ref. 10), and the 02 reduc-tion site (7). Major differences are the electron donor and theabsence of the CUA center from the ubiquinol oxidase (2, 3,6, 9). As suggested here, the only difference in heme structuremay be the replacement of the formyl group of heme A by amethyl group in heme 0. This is expected to lower the Em

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value, which is borne out experimentally (7). Interestingly,the well-conserved tyrosine residue of aa3-type oxidases,which has been implicated in forming a hydrogen bond to theformyl of heme a (35), is replaced by leucine in cytochromeo (2). In bacteria (e.g., Paracoccus denitrificans) that expresscytochromes o and aa3 simultaneously, this difference maybe essential for insertion of the correct prosthetic group.Paracoccus denitrificans membranes indeed contain heme0, based on HPLC analysis (A.P., unpublished results),suggesting that this heme is not unique for cytochrome o fromE. coli.The function of the long hydroxyethylfarnesyl side chain

might simply be to anchor the prosthetic groups tightly to themembranous enzyme, without covalent bonding. But it is notunderstood why this is unnecessary in the cytochrome bc1complex, for example, where the b-type cytochromes containprotoheme (A.P., unpublished results). However, it might berelated to the more complicated chemistry catalyzed by theoxidases, with respect to both 02 reduction and proton-pumping. These functions might require structural transitionsduring the catalytic cycle that necessitate tight anchoring ofthe heme groups.

We thank Hilkka Vuorenmaa for help in preparation of themanuscript and Pentti Somerhaiju for putting his HPLC system atour disposal. This work was supported by grants from the SigridJuselius Foundation and the Academy of Finland.

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