Eur. J. Biochem. 225, 321 -331 (1994) 0 FEBS 1994

Multiple states of the molybdenum centre of dimethylsulphoxide reductase from Rhodobacter capsulatus revealed by EPR spectroscopy Brian BENNETT', Neil BENSON', Alastair G. McEWAN' and Robert C. BRAY' ' Biochemistry Laboratory, School of Chemistry and Molecular Sciences, University of Sussex, Brighton, England

Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Nonvich, England

(Received May 3/July 8, 1994) - EJB 94 0627i4

The dimethylsulphoxide reductase of Rhodobacter capsulatus contains a pterin molybdenum cofactor molecule as its only prosthetic group. Kinetic studies were consistent with re-oxidation of the enzyme being rate limiting in the turnover of dimethylsulphoxide in the presence of the benzyl viologen radical. EPR spectra of molybdenum(V) were generated by reducing the highly purified enzyme under a variety of conditions, and with careful control it was possible to generate at least five clearly distinct EPR signals. These could be simulated, indicating that each corresponds to a single chemical species. Structures of the signal-giving species are discussed in light of the EPR parameters and of information from the literature. Three of the signals show coupling of molybde- num to an exchangeable proton and, in the corresponding species, the metal is presumed to bear a hydroxyl ligand. One signal with g,, 1.96 shows a very strong similarity to a signal for the desulpho form of xanthine oxidase, while two others with g,, values of 1.98 show a distinct similarity to signals from nitrate reductase of Escherichia coli. These data indicate an unusual flexibility in the active site of dimethylsulphoxide reductase, as well as emphasising structural similarities between molybdenum enzymes bearing different forms of the pterin cofactor. Interchange among the different species must involve either a change of coordination geometry, a ligand exchange, or both. The latter may involve replacement of an amino acid residue co-ordinating molybdenum via 0 or N, for a cysteine co-ordinating via S. Since the two signals with g,, 1.96 were obtained only under specific conditions of reduction of the enzyme by dithionite, it is postulated that their generation may be triggered by reduction of the pteridine of the molybdenum cofactor from a dihydro state to the tetrahydro state.

Enzymes that depend on the pterin molybdenum cofactor (Rajagopalan, 1991) are widely distributed, have a variety of important roles in different organisms and have been exten- sively studied in many laboratories (for recent reviews, see Spiro, 1985; Bray, 1988; Solomonson and Barber, 1990; Wootton et al., 1991 ; Enemark and Young, 1993). Detailed structural information from X-ray crystallography is not yet available on any of these enzymes (however, see Romao et al., 1993a,b). All but one of the enzymes contain, in addition to molybdenum, redox centres such as flavin, haem or iron- sulphur. These complicate spectroscopic investigations and, in particular, their strong absorption in the ultraviolethisible region effectively masks the rather weak absorption of the molybdenum chromophore. Nevertheless, other spectro- scopic methods have provided important insights into the lo- cal structure around the molybdenum atom. EPR spectros- copy has been very important in this context and has been supplemented particularly by extended X-ray absorption fine structure (EXAFS) and to a lesser extent by magnetic circular

Correspondence to R. C. Bray, School of Chemistry and Molec- ular Science, University of Sussex, Brighton England BN1 9QJ or A. G. McEwan, Department of Microbiology, University of Queens- land, Brisbane, Australia Qld 4072.

Abbreviations. MCD, magnetic circular dichroism ; EXAFS, ex- tended X-ray absorption fine structure ; Ches, 2-(cyc1ohexamino)- ethanesulphonic acid.

dichroism (MCD) spectroscopy. The special contribution of EPR to molybdenum enzymes has depended on the narrow spectral linewidth shown by molybdenum in the molybde- num(V) oxidation state. Careful examination of the EPR spectra, malung use where appropriate of spectral deconvolu- tion and simulation techniques (Bray and George, 1985; Bray, 1988), has made it clear that the molybdenum centre of each enzyme studied in detail can exist in several different and clearly defined states, each producing its own charcteris- tic molybdenum(V) EPR signal. Much effort has been ex- pended (e.g. George and Bray, 1988; Cleland et al., 1987; Greenwood et al., 1993) in attempting, with the help of model compounds and of isotopic substitution of the en- zymes, to define the precise ligation state and coordination geometry of the metal that gives rise to the individual signals.

The present studies concern the enzyme dimethyl- sulphoxide reductase (Me,SO reductase) from the photosyn- thetic bacterium Rhodobacter capsulatus. This enzyme (McEwan et al., 1991) and the very closely related species from Rhodobacter sphaeroides (Satoh and Kurihara, 1987) both have molybdenum as the sole redox centre, in a protein consisting of a single polypeptide chain of M, approximately 82000. In the latter enzyme, the molybdenum cofactor has been shown to be in a dinucleotide form (Johnson et al., 1991), linked to GMP, and a similar situation has been found for Me,SO reductase from R. capsulatus (N. Benson and A.

322

G. McEwan, unpublished work). The simplicity of Me,SO reductase makes it an attractive model in which to study the structure and function of pterin molybdenum cofactor en- zymes and this has been exploited in two recent studies by MCD spectroscopy (Benson et al., 1992; Finnegan et al., 1993). In the present work, we report the first detailed EPR studies on Me,SO reductase. Analysis of molybdenum(V) signals reveals complex behaviour not apparent in earlier work (Bastian et al., 1991 ; Benson et al., 1992; Finnegan et al., 1993) that may shed further light on outstanding prob- lems concerning this group of enzymes. A preliminary report (Bennett et al., 1994) on parts of this work has been pub- lished.

MATERIALS AND METHODS Enzyme preparation and assay

Me,SO reductase was prepared from R. capsulutus strain H123, grown phototrophically under conditions described by McEwan et al. (1991). Strain H123 is a Tn5 insertion mutant derived from R. capsulutus strain 37b4, used for the purifica- tion of Me,SO reductase in earlier work (McEwan et al., 1991). This mutant lacks a number of c-type cytochromes (McEwan et al., 1989) and this simplified the purification scheme for Me,SO reductase. After preparation of a periplas- mic fraction (McEwan et al., 1991) Me,SO reductase was purified by ammonium sulphate precipitation (50-75 % cut) followed by chromatography on phenyl-Sepharose, as de- scribed in the earlier work.

Examination of M e 3 0 reductase preparations by SDS/ PAGE revealed the presence of a single major polypeptide (Mc = 82000) and analysis of Coomassie-stained gels by la- ser densitometry showed that the purity was >97.5%. N- terminal sequence analysis of the major polypeptide con- firmed that this was a single species. The absence of even trace amounts of contaminating flavoproteins was confirmed by fluorimetric analysis (Burch, 1957) for total flavin. Me,SO reductase was assayed by following the Me,SO-de- pendent oxidation of dithionite-reduced methyl viologen, as described by McEwan et al. (3985). Three batches of enzyme were used in the present work, with specific activities of 45- 50 pmol Me,SO . mg-' . min-' at 30°C. For some experi- ments, enzyme of specific activity 40 pmol . mg-' . min-', that had been recycled by exhaustive dialysis after earlier treatments with reducing and oxidising agents, was em- ployed.

General procedures for generating EPR signals Unless otherwise indicated, all experiments were carried

out in 50 mM Na+-Bicine, pH 8.2, containing 1 mM EDTA. Enzyme samples were concentrated to around 100 pM using Microcon 30 or Minicon B15 concentrators (Amicon Ltd.). Concentrations were estimated spectrophotometrically, by taking E,,,)~,,, = 2 mM-' cm-' (Benson et al., 1992). EPR sample preparation was generally performed in an anaerobic glovebox. Samples were removed in quartz EPR tubes stoppered with rubber seals and frozen immediately in liquid nitrogen. For short incubation times, they were frozen in the glovebox by plunging the tube into a solid COJmethanol bath. For the shortest reaction times, 5 pl reducing agent was introduced with a long-needled syringe, as a drop on the wall near the bottom of the EPR tube (4 mm internal diameter). The enzyme sample (350 pI) was then introduced as a plug

near the top of the tube. The reaction was started by flicking the tube sharply, forcing the enzyme to the bottom and mix- ing it with the reducing agent in one action. Good mixing and times to freezing of a few seconds could be achieved. Samples incubated for successive periods were returned to the glovebox, having been pumped in the air-lock while still frozen. Anaerobic incubations were at 20-25 "C; those car- ried out aerobically for > 30 min were at 4°C. Transfer of enzyme into 'H,O was effected either by dialysis or by buffer exchange using Microcon 30 ultrafiltration units. pD,,, val- ues were measured with a pH meter standardized with 'H,O solutions.

Specific procedures for generating EPR signals and for testing their stability

A variety of treatments were used to generate and test the stability of the different EPR signals from Me,SO reductase. Procedure A corresponds to untreated enzyme, as prepared (or recycled). Reduction with Na,S,O, was used under a vari- ety of conditions, but generally at a final concentration of 1-2.5 mM. Procedures B1, B2 and B3 correspond, respec- tively, to reaction times of < 10 s, 1-5 min and 5-20 min. Procedure B4a corresponds to reduction with dithionite, for up to 2 h, in the presence of the following mediator dyes, each at a concentration of 10 pM, to an apparent indicated potential within the approximate redox buffering range of these relatively high-potential mediators (selected by refer- ence to the work of Bastian et a]., 1991): diaminodurene, 1,2-naphthoquinone, phenazine methosulphate, phenazine ethosulphate, pyocyanine and duroquinone. In other cases (B4b), dithionite was employed at a concentration greater than that of the enzyme plus mediators. Procedure B5 was the same as B3, but the sample was subsequently dialysed anaerobically to remove excess Na,S,O,.

The benzyl viologen radical, BV", was used as reductant in a number of experiments. This was generated electrochem- ically (Thorneley, 1974) and its concentration estimated spectrophotometrically (after anaerobic dilution to minimise the effects of radical dimerization; E ~ ~ ~ ~ , ~ , 7.4 mM-' cm-', Kemp et al., 1975). Procedure C1 corresponds to reduction of the enzyme by approximately 100 pM BV" (i.e. about 1 mol . mol enzyme-') for 1-5 min and C2 to the use of approximately 1 mM BV" for a similar time. Procedure D involved the combined action of BV" and NaBH,. The en- zyme was dialysed anaerobically for 0.5 h against a solution containing 1 mM benzyl viologen, to which solid NaBH, was added until the BV" concentration was approximately 20 pM and the observed potential, relative to the standard hydrogen electrode, approximately -460 mV.

Treatments with oxidizing agents were used either to generate signals from samples reduced (e.g. with Na,S,O,) to the signal-free Mo(1V) state, or to test the stability of signals generated by the procedures described above. E l corresponds to thawing the sample in air and re-freezing after 1-2 min, E2 to exposure to air for 1-20 h and E3 to dialysis in air, to eliminate reductants. Alternatively, oxygen was sometimes employed, and EA corresponds to bubbling oxygen though the solution for < 10 s and E5 to bubbling for 1-5 min. Other oxidants employed were K?Fe(CN), (approximately 1 mM), aerobically for 1-5 min (procedure F1) or for 1 - 20 h, alone (F2), or anaerobically in the presence of media- tors, as in B4 a and b (F3). Finally, Me,SO (approx. 100 mM) was employed aerobically for 1-5 min as oxidant (Gl), or for 1-20 h (G2).

323

I-

8.75 7

EPR spectroscopy EPR spectra were recorded at 9.4 GHz on a Bruker ESP-

300 spectrometer equipped with an NMR gaussmeter (Bruker ER 035 M) and a microwave counter (Marconi Instruments, model 2440). Spectra were transferred to a mainframe com- puter for g-scale alignment, background subtraction, inte- gration and manual or iterative simulation. The software used (G. N. George, unpublished work) was as outlined by Bray and George (1985) and, where employed, difference tech- niques were as summarized by Bray (1988). g-Value esti- mates are believed to be accurate to ?0.0003 or better. Spectra were recorded at 20 mW microwave power, 120 K and 0.2 mT modulation amplitude. Signal acquisition param- eters were chosen such that the spectral resolution was lim- ited by the modulation amplitude. Integrations were com- pared with 2 mM Cu2'-EDTA as standard, with corrections as described in earlier work (e.g. Barber et al., 1976).

a

Pre-steady-state kinetic studies Stopped-flow experiments were carried out using Hi-

Tech equipment (model SF5 I) located inside an anaerobic glovebox. The rapid freezing procedure of Bray (1961) was used as modified in later work (Gutteridge et al., 1978). Sam- ples were frozen 10 ms or 80 ms after mixing the enzyme in one syringe, with a mixture of M e 3 0 and MV'+ (prepared by reduction with substiochiometric Na,S,O,) in another. Fi- nal approximate concentrations were : Me,SO reductase, 60 pM; Me,SO, 60 mM; MV", 600 pM. The buffer was 50 mM Na+-Bicine, pH 8.0, and the temperature 25°C. EPR spectra of these samples showed strong signals from MV", but none from Mo(V). After longer reaction times, as ex- pected, MV" disappeared.

RESULTS Kinetic studies on the nature of the catalytic cycle of Me,SO reductase

We observed a considerable number of different molyb- denum(V) EPR signals from Me,SO reductase. The struc- tures of any molybdenum(V) species that could be shown to be obligatory intermediates in the turnover of the enzyme would be of particular interest. We, therefore, first sought to trap any such species for EPR study by the rapid freezing procedure of Bray (1961), by freezing samples during steady- state turnover of the enzyme in the presence of Me,SO and the methyl viologen radical (MV"), as described in Materi- als and Methods. No molybdenum(V) EPR signals whatever were detected in such experiments. We estimate that signals corresponding to conversion of as little as 0.2% of the en- zyme molybdenum to the molybdenum(\/') state would have been detected. The catalytic cycle is presumably as indicated

Scheme 1. The presumed catalytic cycle of Me,SO reductase. MV"

M O W 7

DMSO

Mo

C e

I I , I I I I

T i m e (s)

I e 8.85 e . 1

Fig. 1. Time course of MV-'disappearance during turnover of Me,SO reductase in the presence of Me,SO and MV.', as ob- served by stopped-flow kinetics. Changes of absorbance (Abs) at 600 nm were followed in 50 mM Na+-Bicine, pH 8.0, at 23"C, with 10 mM Me,SO and approx 60 pM MV" (final concentrations). En- zyme concentrations were 0, 2.5 and 10 pM, respectively, for curves (a), (b) and (c). The experiments were carried out as described in Materials and Methods.

in Scheme 1. Failure to detect molybdenum(V) signals is consistent with relative values of the rate constants being

k, = k2 S k,. (1) That k, is indeed rate limiting is indicated by steady-state kinetic studies, which showed that the catalytic centre activ- ity of Me,SO reductase (calculated by assuming M, 82000) was 62 s-' with Me,SO as oxidant, but 121 s-' or 160 s-', respectively, when the alternative substrates chlorate or trimethylamine N-oxide were used.

If Eqn (1) does apply, an initial burst of MV" consump- tion might be expected to be observable by stopped flow but, as indicated in Fig. 1, we failed to detect such an initial burst. This is probably because the burst was lost in the dead time of the instrument (=3 ms) (As expected, zero-time absor- bance values with 10 pM enzyme were consistently lower, approximately by the predicted extent, than those obtained with buffer only; because of instrument baseline shifts, pre- cise quantitation was not attempted). For the burst to be lost, k , and k2 would have to be close to the diffusion-controlled limit (= 10'M-' s-I), a situation consistent with the very fast reaction of MV" with other proteins, as established by pulse radiolysis (Anderson et al., 1986). The data thus indicate that MV" reacts very rapidly indeed with the molybdenum centre of Me2S0 reductase.

Effects of reductants, oxidants, buffers and other additives on EPR spectra from the enzyme

In general, molybdenum(V) EPR signals might be ex- pected to be generated from Me,SO reductase by stoichio- metric addition of a one-electron reducing agent to the oxi- dized [Mo(VI)] enzyme, or by similar addition of a one- electron oxidizing agent to the fully-reduced [Mo(IV)] en- zyme. Analogous treatment for an appropriate period with an excess of a slow-acting one-electron oxidizing or reducing agent should also yield to the Mo(V) state, as should also experiments at defined redox potentials. We have used most of these approaches to generate EPR signals from M e 3 0 reductase, as is detailed in Materials and Methods. Note that (as discussed above) MV" (and no doubt also BV") is a very rapidly reacting one-electron reducing agent for the en-

3 24

zyme, whereas dithionite, of which we made extensive use, acts on many proteins as a slow one-electron reductant (Lam- beth and Palmer, 1973). Data presented below indicate that oxygen can act as a slow one-electron oxidant for the en- zyme. Most of our experiments were carried out in 50 mM Na+-Bicine, pH 8.2, however, some work was performed at other pH values and in the presence of additives.

EPR spectra that we obtained from Me,SO reductase are illustrated in Figs 2-4. Computer simulations were used for extraction of the EPR parameters (Table 1) and to establish (except where otherwise indicated) that all the signals are due to single chemical species. Note that, in a few cases, as indicated in the figure captions, difference spectral pro- cedures were employed to obtain the spectra illustrated. All the signals presented have been obtained consistently from more than one batch of the enzyme.

In Table 1, signals from Me,SO reductase have been di- vided into four groups. The nomenclature used is as follows. The unique signal obtained in the presence of NaBH, is termed the Borohydride signal. Other signals fall clearly into those with g,, approximately 1.98, termed High-g signals and those with g,, approximately 1.96, termed Low-g signals. The former signals then fall into two classes, depending on whether or not splitting due to interaction with a single strongly coupled proton, is observed. These are termed High- g Split and High-g Unsplit, respectively. Within these main groups, further subdivision is indicated by the suffix type-1 or type-2, or by adding, in parentheses, an indication of the conditions under which the signal was generated.

Previous investigations of Me,SO reductase have re- vealed only the High-g signals. We, therefore, consider these first.

The High-g Split signal The High-g Split signal is the one most readily obtainable

from Me,SO reductase and our results relating to it are analo- gous to the less detailed studies of earlier workers (Bastian et al., 1991; Benson et a]., 1992). We generated the signal by a variety of procedures, including limited reduction with dithionite (procedures B1 or B5 ; see Materials and Methods), or BV" (procedure C1) and more extensive reduction to the EPR-silent state, followed by limited re-oxidation (pro- cedures B3 + E5 or B4 + F3). Conversion to the signal- giving state, in different experiments, ranged over 5 -25% of the molybdenum content. The signal disappeared only slowly on treatment with air (procedures E2 or E3) but more rapidly with oxygen (E5) or Me,SO (Gl). It was readily abolished by further reduction (B3 or C2).

Representative spectra are illustrated in Fig. 2 and param- eters are given in Table 1. Experiments in 'H,O provided more rigorous confirmation of earlier conclusions (Bastian et al., 1991) that a single proton, exchangeable with the solvent and strongly coupled to molybdenum, is responsible for the observed splitting. Precise parameters of the signal (Table 1, Fig. 2) depended critically but reproducibly, on the medium in which it was generated. By analogy with Escherichia coli nitrate reductase (George et al., 1985, 1989a,b) this is most likely due to the co-ordination, in the signal-giving species, of buffer ions or other anions to the molybdenum atom. Fig. 2a and h show the High-g Split signal developed, respec- tively, in Mes, pH 5.6, and in Bicine, pH 8.2 with added gly- col. (The rationale for addition of the latter is explained be- low). The quality of the simulations (Fig. 2b and i) is indica- tive of single species and there is a clearly significant shift

2.01 2.00 1.39 1.38 1.97 1.96 1.95

g value

Fig. 2. The High-g Split EPR signals from R. capsulatus Me,SO reductase. Experimental spectra are shown as fuL2 lines in (a), (c), (d), (e) ( f ) , (g), (h), (i) and (1) and computer simulations, obtained by using the parameters given in Table 1, are shown as dashed lines in (b), (i), (k) and (m), corresponding, respectively to (a), (h), (i) and (I). Spectra (i) and (I) are difference spectra obtained by appropriate subtraction of (f) from (g) and back subtraction of (j) from (f), re- spectively [High-g Split (Bicine) type-1 and type-2 signals; 'H,O form]. All spectra are shown normalized to a common amplitude and were generated by using procedures described in Materials and Methods. The sample for (a) was reduced by procedure B5 in Na'Mes, pH 5.6, [High-g Split (Mes) signal], that for (c) by pro- cedures B3 + E5 in Na' Ches, pH 9.0, that for (d) by procedure C1 (note that the feature centered at approximately g 2.003 presumably corresponds to BV"), that for (e) by procedures B4 + F3. Spectra ( f ) and (g) both correspond to samples in *H,O reduced by procedure B1 for nominal reaction times of 2 s and 5 s, respectively. Sample (h) was prepared in the presence of 10% (by vol.) ethylene glycol by procedures B4b followed by procedures E5 [High-g Split (gly- col)] signal.

of 0.0014 in g,, between the two species (Table 1). Compari- son of these spectra with those given in Fig. 2c, d and e indicates further small but significant changes, but the lines- hape suggests in each of these cases that more than one spe- cies is involved and simulations are, therefore, not shown. In fact, the lineshape in Fig. 2e is simulatable (data not shown) in terms of a single species bearing a strongly coupled and a more weakly coupled proton, as is suggested by the 'double doublet' apparent in the g, region. However, this spectrum may also be simulated (data not shown) in terms of two spe- cies having slightly different parameters and present in equal amounts. That this is the true explanation is shown by exami-

325

nation, by difference spectral techniques, of the spectra (Fig. 2f and g of a closely related pair of samples, analogous to that in Fig. 2e but prepared in ,H,O and, therefore, lacking the splitting from the strongly coupled proton. This permitted these spectra to be resolved into the two species, shown in Fig. 2j and 1 and simulated in Fig. 2k and m, respectively. We refer to these as the High-g Split (Bicine) signals, types 1 and 2, respectively. In fact, the parameters (Table 1) from the simulation of Fig. 2k are indistinguishable, within experi- mental error, from those in Fig. 2i (though, of course, the proton coupling could not be reliably estimated from the for- mer). A plausible explanation would be that the types 1 and 2 signals correspond to the co-ordination to molybdenum of, in one case, the Bicine anion, and in the other, the Bicine zwitterion. The action of glycol might then be simply to fa- vour one of these complexes over the other.

Parameters of all forms of the High-g Split signal are quite similar (Table 1) to those of the Low-pH signal from E. coli nitrate reductase, most particularly to the nitrite type- 1 form of this signal (George et al., 1985). However, in sharp contrast to the situation with that enzyme, we find no evi- dence whatever for a pH-dependent equilibrium between this EPR signal-giving form of Me,SO reductase and another form lacking a strongly coupled proton. Thus, although in Fig. 2 different spectra were recorded at pH values ranging over 5.6-9.0, differences amongst the spectra appear to be due, as already discussed, to complex formation with buffer constituents rather than to generalized pH effects.

In agreement with Bastian et al. (1991), in samples at pH values above 10, a very weak new signal appeared, appar- ently related to degradation of the enzyme. We did not study this signal further.

The High-g Unsplit signal

Signals in this category have been reported by Finnegan et al. (1993) and by Benson et al. (1992) for Me,SO reduc- tase from Rhodobacter, and by Cammack and Weiner (1990) for the structurally distinct Me,SO reductase of E. coli. Our data on three forms of the signal are summarized in Fig. 3 and Table 1. The High-g Unsplit type-1 signal (Fig. 3a) was sometimes observed, in agreement with Benson et al. (1992), in untreated samples of the enzyme (procedure A). The sig- nal was always weak (<2.5% of the total Mo content), but we were not able to confirm the suggestion of these workers that its presence was confined to enzyme samples of low specific activity. The signal was not changed in form by dia- lysing the sample, originally in 50 mM Bicine, pH 8.2, into Mes, pH 5.6, or 2-(cyclohexamino)ethanesulphonic acid (Ches), pH 9.0. The type-2 variant of the signal (Fig. 3c) was generated at an intensity corresponding to up to 5% of the molybdenum, on reducing samples of the enzyme, then re- oxidizing them (procedures B4 and F3 2 Gl). Our work on the High-g Unsplit (glycol) signal is related to the studies of Finnegan et al. (1993), who reported that, on reducing Me,SO reductase from R. sphaeroides with BV", then add- ing 50% (by vol.) glycerol, 80% of the molybdenum was converted to a species giving an unsplit signal with the g values quoted in Table 1. Signal development was reported to be accompanied by complete loss of enzymic activity. For the R. capsulatus enzyme, we used 10% (by vol.) glycol in place of glycerol. Reduction in this medium by procedure B3 followed by re-oxidation (E4), as already noted, gave the High-g Split signal. However, on extending the oxidative treatment (E5+E2), this signal was replaced by the High-g

I I I I I I

2.00 1.99 1.98 1.37 1.36 1.95

g value

Fig.3. The High-g Unsplit and Borohydride EPR signals from R. cupsulutus Me,SO reductase. Experimental spectra are shown as full lines in (a), (c), (e) and (8) and the corresponding computer simulations obtained by using the parameters given in Table 1, as dashed lines in (b), (d), (f) and (h), respectively. Spectra were gener- ated by using procedures described in Materials and Methods. The sample in (a) was generated by procedure A (High-g Unsplit type- 1 signal), that in (c ) by procedures B4 and F3 (High-g Unsplit type- 2 signal; note that a small amount of the High-g Split signal has been subtracted from the spectrum shown), that in (e) in the presence of 10% (by vol.) ethylene glycol by procedures B4b and E5 (Fig. 2h) + E2 [High-g Unsplit (glycol) signal], and that in (g) by procedure D (Borohydride signal).

Unsplit (glycol) signal (Fig. 3e), with parameters (Table 1) indistinguishable from those described by Finnegan et al. (1993) (within the limits of accuracy quoted). However, in contrast to these workers, we found no loss of enzymic activ- ity and only approximately 7 % conversion to the signal-giv- ing species.

We found all three forms of the High-g Unsplit signal to be resistant to oxidative treatment with oxygen or M e 3 0 (procedures E5 or Gl). Indeed, oxidation of the other Mo(V) EPR signal-giving species with Me,SO could be used to ob- tain the High-g Unsplit signal in isolation. However, and in contrast to the analogous work of Finnegan et al. (1993), the High-g Unsplit (glycol) signal disappeared after exhaustive dialysis (E3). All three forms disappeared on further reduc- tion of the samples with dithionite (B3) or BV" (C2) and did not re-appear on subsequent aerobic dialysis (E3).

The High-g Unsplit signal, in all its forms, is quite simi- lar to the High-pH signal (George et al., 1985) for E. coli nitrate reductase (see Table 1). However, there is an impor- tant difference. Whereas the latter shows weak coupling of an exchangeable proton to molybdenum, with A, (H)av 0.34 mT, we were able to detect no change in EPR linewidth either directly, or by the use of second-derivative spectra, on exchanging a sample giving the High-g Unsplit type-1 signal into 'H,O.

The Borohydride signal

In searching for alternative reductants of the enzyme, we observed a unique signal referred to as the Borohydride sig-

- m Q Q aa h 3

nal (Fig. 3g; Table 1). We generated it only by use of BV" in the presence of NaBH, (procedure D) and were unable to replace this system by other reductants, such as dithionite in the presence of borate ions. The signal shows no evidence, even in higher derivative spectra, for splittings due to hyper- fine coupling to protons or to the "B nucleus, and is unique in signals from the enzyme in having gll > g,. Conversion to the signal-giving species varied in different experiments from 2-30% molybdenum and was always accompanied, even when the conversion was low, by some loss of enzymic activity, e.g. 40% loss. The signal was more stable than oth- ers from the enzyme. Thus, it was resistant to oxidation by oxygen (E5) or Me,SO (Gl), though it did disappear on pro- longed dialysis (E3). On addition of dithionite (B3) the signal disappeared, to be replaced by additional transient signals that we did not study in detail. On subsequent reoxidation of the fully reduced enzyme with oxygen (E5) or M e 3 0 (G2), the original Borohydride signal re-appeared.

The Low-g type-1 and type-2 signals

4- '3 05 =,

- _ - !? Z L $ e E

.5

signal (see Discus&) and of its relatively low intensity, it was particularly important to confirm as rigorously as pos- sible (see Materials and Methods) that the signal was due to Me,SO reductase molecules and not to any possible contami-

>

Two new signals not previously described for Me,SO re- ductase were generated on reduction of the enzyme under specific conditions with dithionite. These are illustrated in Fig. 4, and they are characterised by g,, values of approxi- mately 1.96 (Table 1). The Low-g type-1 signal (Fig. 4b and d) shows splitting from a single exchangeable proton. It bears a striking similarity (Table 1) to a signal (Fig. 4a) from the desulpho form of xanthine oxidase, known as the Slow sig- nal, and in particular to the variant of this obtained in the presence of nitrate ions (Gutteridge et al., 1978). The Low-g type-2 signal (Fig. 4f and h) is also split by an exchangeable proton and has an even lower value of g,, than the type-1 signal, and its g values are much more anisotropic (Table 1).

We succeeded in generating the Low-g signals only in Bicine, pH 8.2, when using dithionite as reductant. Moder- ately high concentrations of the reductant were employed with intermediate reaction times (procedures B2, B3 or B4). Appearance of the Low-g type-1 signal was sometimes pre- ceded by transient appearance of the High-g Split signal, and that of the Low-g type-2 signal, by transient appearance of the type-1 signal. Both signals were obtained from more than one preparation of the enzyme, but conversions to the signal- giving species in different experiments were somewhat vari- able, ranging over 0.8-5% of the molybdenum for the type- 1 signal and up to 12% for the type-2 signal. Both signals disappeared on extended treatment with dithionite in the ab- sence of additives (B3). On oxidative treatment (E4) of such samples, the type-1 but not the type-2 signal has been ob- served. Attempts to generate the Low-g signals in Mes, pH 5.6, resulted in the appearance of additional signals, as did also the extended reduction by dithionite in Bicine, pH 8.2, in the presence of mediator dyes. We did not study these signals in detail. No analogous signals were observed in Ches, pH 9.0. Attempts to generate the Low-g signals in Bicine, pH 8.2, using other reductants in place of dithionite, were entirely unsuccessful. Those tried included NaBH,, and BV' + or MV", generated electrochemically; in some experi- ments NaHSO, or Na,S,O, oxidation products were added, but without success.

In view of the importance we attach to the Low-n type-1

327

I I I i 1 I I I

1.99 1.98 1.97 1.96 1.95 1.94 1.93 1.92

g value Fig. 4. The Low-g EPR signals from R. capsulatus Me,SO reduc- tase and comparison with the Slow signal from desulpho xan- thine oxidase. Experimental spectra from Me,SO reductase are shown as full lines in (b), (d), (f) and (h) and the corresponding computer simulations obtained by using the parameters given in Table 1, as dashed lines in (c), (e), (g) and (i), respectively. The simulation of the Slow (nitrate) signal from desulpho xanthine oxi- dase (Gutteridge et al., 1978) is presented in (a) for comparison. Spectra were generated using procedures described in Materials and Methods. The sample for (b) was reduced by procedure B4 (Low-g type-1 signal; note that a small other signal obtained under slightly different reaction conditions in the same experiment has been subtracted from the spectrum shown), that in (d) in 2H,0 by pro- cedure B2 (Low-g type-I signal), that in (f) by procedure B1 + B2 (Low-g type-2 signal) and that in (h) in 'H,O by procedures B1 (Fig. 2f) + B3 [Low-g type-2 signal].

nating molybdo proteins present in the samples, and in par- ticular not to a molybdenum-containing hydroxylase in the desulpho form. Such enzymes are not known to be present in R. cupsulutus. In support of their absence, such trace im- purity bands as we observed in SDS/polyacrylamide gel elec- trophoresis had lower rather than higher M, values than the main band. Subunit M, values for non-proteolysed forms of molybdenum-containing hydroxylases (Keith et al., 1987 ; Romao et al., 1993a) are higher than that of for R. cupsulutus Me,SO reductase. Furthermore, even in what is clearly the unlikely event that some contaminant of Me,SO reductase, present at the limit of 2.5% the concentration of the enzyme, was, in fact, the desulpho form of a molybdenum-containing hydroxylase, no more than a fraction of the molybdenum would be converted to the signal-giving species. This clearly excludes the possibility of all but the weakest of the observed Low-g type-1 signals from being derived from such impuri- ties. As a final check on the absence of a flavin-containing molybdenum-containing hydroxylase, we submitted a sample of the enzyme that had yielded a rather weak Low-g type-1 signal corresponding to 0.8% of the molybdenum present, to fluorimetric flavin analysis. The upper limit found for any

possible flavin contamination was considerably lower than the signal intensity, corresponding, on a molar basis, to no more than 0.04% of the molybdenum content. Clearly, all the evidence points to all the Mo(V) EPR signals that we ob- served having originated from Me,SO reductase molecules.

DISCUSSION General structural inferences from the EPR parameters

Me,SO reductase from R. cupsulutus is unusual amongst molybdenum enzymes in the large number of distinct molyb- denum(V) EPR signals that it gives. Molybdenum-containing hydroxylases, typified by milk xanthine oxidase, give rise to a comparable number of signals, but for these enzymes some signals arise not from the functional form but from the con- taminating desulpho modification. More typically, E. coli ni- trate reductase and liver sulphite oxidase give rise to only two or three well-defined signals each. The large number of signals must imply structural flexibility in the molybdenum centre of Me,SO reductase, permitting it to adopt a corre- sponding number of different conformations. Most signifi- cantly, the molybdenum centre of this enzyme with the metal in the Mo(V) oxidation state, can adopt conformations typi- cal of what were hitherto regarded as two different families (Wootton et al., 1991) of molybdenum enzymes. Thus, when producing Low-g type-1 signal, its conformation must be re- markably similar to that of desulpho xanthine oxidase, when this produces the Slow signal. In contrast, when the enzyme produces the High-g signals, it must be similar to that of E. coli nitrate reductase. We have given careful consideration to the possibility that the Low-g type-1 signal arises from an impurity in our enzyme preparations but the data are clearly not consistent with this. It is particularly significant that while xanthine oxidase contains the molybdenum cofactor as the simple molybdopterin, in E. coli nitrate reductase and M e 3 0 redutase the molybdenum cofactor is present as the dinucleotide with GMP (Rajagoplan, 1991). Thus, the struc- ture round molybdenum does not appear to be uniquely influ- enced by the presence of the nucleotide. This is perhaps not surprising in view of the extended cofactor structure indi- cated in xanthine oxidase and sulphite oxidase by 31P electron nuclear double-resonance studies (Howes et al., 1991).

In considering the structure round the molybdenum atom in the different signal-giving species, we start with the Slow signal from desulpho xanthine oxidase. The most likely structure for this species, based on EPR and EXAFS data, is shown in Scheme 2a (Wootton et al., 1991; Turner et al., 1989; Bray, 1988). In view of the remarkable similarity (Table 1) of the parameters of this signal to those of the Low-g type-1 signal from Me,SO reductase, we infer that the structure illustrated applies also to this enzyme, when in this signal-giving state. Proceeding to the structures of other sig- nal-giving species from Me,SO reductase, it seems certain in the light of available EPR and EXAFS data on other molyb- denum enzymes, that the 0x0 (=O) ligand is retained in all of the enzymes, with the hydroxyl group retained as the source of the strongly coupled, solvent-exchangeable proton in all the species that show proton splitting. Though there are quite large differences in the parameters of the different sig- nals, which undoubtedly have their origins in defined struc- tural changes, nevertheless, drawing specific structural con- clusions is not easy. As is emphasised by the work of Cleland et al. (1987) on a unique series molybdenum(V) model com- pounds, EPR parameters are extremely sensitive to changes

328

Table 2. Effect of structural changes on Mo(V) EPR parameters of model compounds and of some molybdoenzyme species. EPR parameters for various molybdenum(V) model compounds from the literature are listed, with those for different signals from Me,SO reductase (from Table 1) and for signals from other enzymes. In italics, on lines intervening between pairs of data sets, the change in the direction indicated by the arrow, in each of the parameters listed is given, together with the nature of the structural change, where this is known. L = hydrotris (3,5-dimethyl-l-pyrazolyl) borate; enzymes: XO, xanthine oxidase; XO(ds), xanthine oxidase in the desulpho form, SO, liver sulphite oxidase; DMSOR, Me,SO reductase from R. capsulatus. Anisotropy and rhombicity parameters are as defined in Table 1.

Compound Structural change ga" Anisotropy Rhombicity Reference or enzymehignal

XO(ds)/Slow (Bicine) 1.9647 0.0168 0.29 Gutteridge et al. (1 978)

XO/Rapid (formamide) 1.9759 0.0235 0.81 George and Bray (1988) LMoO(OMe), 1.935 0.056 0.32 Cleland et al. (1987)

LMoO(OCH,CH,O) 1.948 0.066 0.17 Cleland et al. (1987)

LMoO(OCH,CH,S) 1.963 0.059 0.44 Cleland et al. (1987) LMoO(SEt), 1.965 0.080 0.74 Cleland et al. (1987)

LMoO(SCH,CH,S) 1.976 0.074 0.61 Cleland et al. (1987) SO/High pH 1.9679 0.0342 0.69 George et al. (1985)

SOLow pH (chloride) 1.9805 0.0379 0.84 Lamy et al. (1980) DMSORLOW-g 1.9647 0.0142 0.17 present work

DMSOR/High-g Split (Bicine) 1.9816 0.252 0.42 present work

DMSOlULow-g type 2 1.9553 0.0400 0.55 present work

1 04s +0.011 + 0.008 +0.52

1 chelate effect +0.013 +O.OlO -0.15

1 04s f0.015 -0.007 +0.27

1 chelate effect +0.011 - 0.006 -0.13

1 ligand replaced ( ?) +0.013 + 0.004 +O.15

1 ? f0.017 +0.011 + 0.25

T ? +0.026 -0.015 -0.13

in the donor atoms and to the geometrical constraints of the co-ordinated ligands. This makes it difficult to distinguish between changes in ligand atom and even small changes of coordination geometry.

The relevant thoery is well understood at the qualitative level (Enemark and Young, 1993) but quantitative experi- mental information is more helpful. In Table 2, data on the magnitude of changes in the EPR parameters produced by defined structural alternations in a series of model com- pounds are compared with corresponding data on various molybdenum enzymes and with the present data on Me,SO reductase. Values of g,, and of the rhombicity and anisotropy parameters are listed. Replacement of an oxygen ligand by sulphur, as when LMoO(OCH,CH,O) is compared with LMoO(OCH,CH,S), gives rise to increases in both g,, and the rhombicity parameter and, indeed, this holds also for re- placement of either oxygen or nitrogen by sulphur, in each of eight pairs of derivatives of the ligand, L [hydrotris(3,5- dimethyl-1 -pyrazolyl)borate], studied by Cleland et al. (1987). The changes in parameters are comparable (Table 2), when the Slow signal from desulpho xanthine oxidase is compared with the Rapid signal from this enzyme in the functional state. The corresponding structural change is pre- sumed (Bray, 1988) to involve replacement of an -OH by an -SH ligand. Also comparable, are the parameters changes when the High-pH and Low-pH species from sulphite oxi- dase are considered, though here, in contrast, there is no in- formation available other than the EPR parameters, support- ing such a change of ligand (see George et al., 1989). In contrast, the work of Cleland et al. (1987) shows that compa- rable g,, increases are also produced by quite subtle changes of geometry, without a change of ligand type, as when two sulphur or oxygen ligands in separate co-ordinated molecules are replaced by the same two ligands contained within a sin- gle molecule (Table 2, chelate effect). In the two pairs of

compounds cited in Table 2, as well as in other pairs given by these workers, the increased g,, arising from the chelate effect is accompanied by a decrease in the rhombicity param- eter.

It is hazardous to extrapolate from these data derived from model compounds having a particular geometry, to en- zymes with no doubt different geometries. Nevertheless, the data provide a background against which those relating to Me,SO reductase may be considered. It is immediately clear that the change from Low-g type-1 species to High-g Split would be consistent with a change to ligation by sulphur, while a change of geometry might be sufficient to explain the change from the latter species to Low-g type-2.

Possible structures of the various signal-giving species from Me,SO reductase

Based on the considerations given above, we tentatively put forward the structures shown in Scheme 2 for the main signal-giving species from Me,SO reductase. The difference between the Low-g type-1 and the High-g Split species (Scheme 2a and c) is shown as due to replacement of an unspecified amino acid coordinating through oxygen or ni- trogen in the former, by a thiolate ligand from a cysteine residue in the latter. While low-temperature MCD (Benson et al., 1992) provided no evidence supporting such a ligand in this species, the presence of such a ligand was not categor- ically excluded by these workers. The difference between the type-2 and the type-1 Low-g species (Scheme 2b and a) is shown as a conformation change, symbolised by the inter- change of the ligands B and OH. The ligand X, that in the High-g Unsplit species replaces the hydroxyl ligand respon- sible for the proton splittings, may, in the glycol form of the signal, be from a glycol molecule. However, in contrast to the conclusion of Finnegan et al. (1993), we suggest that this

3 29

Scheme 2. Possible co-ordination of Mo(V) in different signal- giving species from Me,SO reductase. The structures shown are suggested to correspond to: (a) the Low-g type-I species, as well as the Slow species from desulpho xanthine oxidase, (b) the Low-g type-2 species, (c) the High-g Split species and (d) the High-g Un- split species. B represents an anion ligand, Such as a buffer ion; AA is an unspecified ligand from an amino acid side chain in the protein, replaced by some species by Cys, which is a thiolate ligand from a cysteine residue. X is an unspecified ligand, possibly from the pro- tein or from the medium. The two sulphur atoms of the cofactor dithiolene system are shown joined by a curved line. This line is represented differently in the Low-g and High-g species, to indicate a possible difference in cofactor conformation, perhaps related to the oxidation state of the pterin. (Note that effects of such a change might be sufficient to explain, on their own, the difference between the structures of these species, without the postulate of a change of amino acid ligand).

a b

B\Mo/ 0 cys

/ : \ HO j S

S d

C d

is bound in a monodentate rather than a bidentate manner. The nature of X in the High-g Unsplit type-1 and type-2 signals is uncertain. The nature of B, as a buffer ion or anion, has already been alluded to in relation to the High-g Split signal. Similarly, there is evidence (Gutteridge et al., 1978; Bray, 1988) for such a ligand in the Slow species from desul- pho xanthine oxidase and, hence, by analogy also in the Low-g type-1 signal from Me,SO reductase.

We hesitate to speculate on the structure of the Borohy- dride signal-giving species but it may well be that, despite the absence of detectable hyperfine coupling to boron, the boron atom remains bound. Conceivably, reaction could oc- cur both at the molybdenum and at the free hydroxyl group in the 3' position of the pteridine side chain, with the boron atom bridging between. Model building has shown the feasi- bility of such a structure.

Origins of the structural differences between the High-g and Low-g signals

It seems significant that the Low-g signals were gener- ated only by the action of dithionite and, notwithstanding the structures put forward in the preceding subsection, it is conceivable that this anion might be coordinated to molybde- num, perhaps in a manner analgous to that in which the bisul- phite ion binds tightly (Fish et al., 1990) to the metal in xanthine oxidase. However, this possibility can be dis- counted, at least in the case of the Low-g type-1 species, in view of the similarity of fhe signal fo the Slow signal from desulpho xanthine oxidase which, i t is known (Swann and Bray, 1972), can be generated by reductants other than di-

thionite. Another possibility might be that sulphite or bisul- phite ions from dithionite oxidation are involved in the struc- tures of the Low-g species, but against this is our failure to obtain the signals, e.g. with sulphite and BV".

It seems, therefore, that we must look to the reducing powers of dithionite to explain the unique ability of this rea- gent to generate the Low-g signals. Reductions by dithionite are complicated (Lambeth and Palmer, 1973 ; Mayhew, 1978) but at low concentrations, at pH 8.2, the SO;-/HSO; couple has a potential near to -800 mV, some 300 mV more nega- tive than that of the methyl violgen couple (Mayhew, 1978). Dithionite is also known (Kawai and Scrimgeour, 1972) to be capable, if sometimes in low yield, of reducing free pteri- dines to the tetrahydro state. It is, therefore, tempting to sug- gest that the basic difference between the High-g and the Low-g species arises from changes in the redox state of the pteridine ring system of the molybdenum cofactor. Some de- viations from the planar structure of the oxidized pteridine molecule are expected (Pfleiderer, 1985) on reduction to the tetrahydro form, and such changes could well have a substan- tial effect on the environment of the molybdenum, perhaps triggering the ligation changes indicated in Scheme 2.

Gardlik and Rajagopalan (1990, 1991) made strenuous efforts to determine the redox state of the pteridine in xan- thine oxidase and sulphite oxidase. Their conclusion was that, in both cases, a dihydropteridine was involved, but with a different isomer in the two enzymes. These experiments are technically difficult and the reported results contain certain anomalies. In the absence of precautions to prevent a change of cofactor redox state after liberation, Gardlik and Ragago- palan (1990) reported that the tetrahydro form was liberated on denaturation of xanthine oxidase, while sulphite oxidase gave a dihydro form. Attempts by these workers to use mer- curic ions to prevent the postulated cofactor reduction by sulphide ions, arising from the sulphido ligand of molybde- num in xanthine oxidase, were only partly successful. Furthermore, their conclusion of a correlation between the oxidation state of the liberated pteridine and the relative amounts of the functional and desulpho enzyme forms in the xanthine oxidase preparations employed, is seriously marred if their data on cyanide-treated enzyme are included. Thus, some distinct support may, in fact, be inferred from the work of Gardlik and Rajagopalan (1990) for the possibility that the pteridine is normally in the tetrahydro state in xanthine oxi- dase and the dihydro state in sulphite oxidase.

It is tempting to postulate, therefore, that the molybde- num centre of dimethylsulphoxide reductase, normally in the High-g, nitrate-reductase-like conformation, can adopt a de- sulpho xanthine-oxidase-like conformation on reduction by dithionite of the pteridine from the dihydro to the tetrahydro form, as in the Low-g type-1 species. Such a proposal has important implications concerning possible roles (Rajagoplan 1991 ; Russell et al., 1992) of the pteridine in the cofactor. In particular, it would be consistent with the suggestion (one of the many put forward by Rajagopalan, 1991) that the pterin oxidation state might control the redox potentials of molyb- denum in molybdoenzymes. Further work will be required to test our hypothesis. In particular, it will be necessary to de- fine more precisely the conditions required to achieve the postulated full reduction of the pterin of Me,SO reductase, while retaining its molybdenum in the Mo(V) state. Clearly, from the relatively elusive nature of the Low-g signals found in the present work and from the failure of earlier workers to detect them, the range of conditions under which this might be achievable must be rather limited.

3 30

Relations of the different signal-giving species to Me,SO reductase function

Although we did not prove the matter rigorously by pre- steady kinetic studies, the most likely candidate amongst the various molybdenum(V) species for that participating in the catalytic cycle according to Scheme 1, is the High-g Split species. This signal was readily generated by a variety of reductants, including BV”, and disappeared readily on oxi- dation, e.g. by Me,SO. The relationship of other species to the catalytic cycle is more problematical. While there is no definitive information in our work linking the High-g Unsplit species to ‘dead’ or ‘temporarily dead’ enzyme molecules, such a relationship is a possibility. These signals, though rel- atively resistant to oxidation, were eliminated by reducing the enzyme to the Mo (IV) state, followed by re-oxidation. As already noted, our data relating to the glycol form of this signal differ importantly from those reported by Finnegan et al. (1993) for R. sphaeroides Me,SO reductase treated with glycerol, though the signals themselves appear identical. Our findings of low conversion to the signal-giving species, re- tention of enzymic activity and loss of the signal on oxida- tion, all weaken the conclusion of these workers of a struc- tural analogy to the very stable Desulpho-Inhibited species (Bray, 1988) for desulpho xanthine oxidase.

Our failure to generate the Low-g signals with reductants other than dithionite makes it quite unlikely that the signal- giving species are related directly to enzyme forms partici- pating in the catalytic cycle. In contrast to these, the Borohy- dride signal was particularly stable and is most likely due to an inactivated derivative of the enzyme.

We thank Prof. A. J. Thomson, FRS, for helpful discussions, Mr Richard Little for help with the preparation of Me,SO reductase, Ms Gill Ashby for skilled assistance with the stopped-flow equipment, Dr R. N. F. Thorneley for making this available to us and the Univer- sity of Brighton Computing Centre for the use of their VAX 8700 computer. The work was supported by grants to R. C. B. from the AFRC (Linked Research Group with the Nitrogen Fixation Labora- tory) and from the SERC (project grant and grant for purchase of the EPR spectrometer, on which all the work described was carried out) and to A. G. McE. from the Science and Engineering Research Council (project grant and support for the Centre for Metalloprotein Spectroscopy). R. C. B. thanks the Leverhulme Trust for an Emeri- tus Fellowship.

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