Spectroscopic studies of the oxidation-reduction properties of three forms of ferredoxin from Desulphovibrio gigas

Biochimica et Biophysica Acta, 490 (1977) 3l 1-321

© Elsevier/North-Holland Biomedical Press BBA 37557 SPECTROSCOPIC STUDIES OF T H E O X I D A T I O N - R E D U C T I O N PROPERTIES OF T H R E E F O R M S OF F E R R E D O X I N F R O M D E S U L P H O V I B R I O GIGAS

RICHARD CAMMACK', K. KRISHNA RAOa, DAVID O. HALLa, JOSl~ J. G. MOURA b, ANTONIO V. XAVIER b, MIREILLE BRUSCHI c, JEAN LE GALLc, ALAIN DEVILLEd and JEAN-PIERRE GAYDA d aDepartment of Plant Sciences, University of London, Kings College, 68 Half Moon Lane, London SE24 9JF (U.K.), bCentro da Quimica Estrl~tural da Universidade de Lisboa, 1.S.T., Lisbon 1 (Portugal), ~Laboratoire de Chimie Bactdridnne, CNRS, 13274 Marseille C~dex 2, and dDepartment of Electronics, Universitd de Provence, St. J~rome, 13397 Marseille, C~dex 4 (France)

(Received July 29th, 1976)

SUMMARY Electron paramagnetic resonance spectra were recorded of three forms of Desulphovibrio gigas ferredoxin, FdI, FdI' and FdlI. The g---- 1.94 signal seen in

dithionite-reduced samples is strong in FdI, weaker in FdI' and very small in FdlI. The g = 2.02 signal in the oxidized proteins is weak in FdI and strongest in FdlI. It is concluded that most of the 4Fe-4S centres in FdI change between the oxidation states C 2- and C 3-, while those in FdlI change between states C - and C2-; FdI' contains both types of centre. There is no evidence that any particular centre can change reversibly between all three oxidation states. Circular dichroism spectra show differences between FdI and FdlI even in the diamagnetic C z- state. The redox potentials of the iron-sulphur centres of the three oligomers (forms) are different. After formation of the apo-protein of FdlI and reconstitution with iron and sulphide, the protein behaves more like FdI, showing a strong g ---- 1.94 signal in the reduced state.

INTRODUCTION A previous paper [1] has reported the isolation and some properties of three ferredoxin fractions, termed FdI, FdI' and FdlI, from the sulphate reducing bacterium Desulphovibrio gigas. The molecular weights of these oligomeric forms are 18 000, 18 000 and 24 000 respectively. On the basis of amino acid composition they all appear to be polymers of a basic subunit of molecular weight approx. 6000, containing one 4Se-4S duster. The amino acid sequence of the subunit protein has been determined [2]. Magnetic susceptibility and contact-shifted proton nuclear magnetic resonance (Moura, J. J. G., Xavier, A. V., Bruschi, M., Le Gall, J., Biochim. Biophys. Acta., in press) show that FdI resembles typical bacterial ferredoxins in that it has only residual paramagnetism in the oxidized state, with an optical absorption maximum

312 at 405 nm. On reduction with dithionite, the molecule becomes paramagnetic. This form represents the C 3- state of the iron-sulphur cluster, in the "three-state" terminology of Carter et al. [3]. Its visible absorption is considerably decreased. FdlI on the other hand, is paramagnetic in its oxidized form, as prepared, and has an optical absorption maximum at 415 nm. The reduced form has a lower magnetic susceptibility and shows an absorption band around 425 nm [1]. In these properties it resembles the high-potential iron-sulphur protein (HiP1P) from Chromatium. The latter protein contains a 4Fe-4S cluster similar to those in the bacterial ferredoxins, but has a much higher redox potential (T350 mV) and an EPR signal, with g-values greater than 2, in the oxidized state rather than the reduced state [4, 5]. FdI' is intermediate in properties between I and II, although it is a distinct protein fraction. The properties of the three forms of the ferredoxin can therefore be rationalized in terms of three oxidation states, i.e. the paramagnetic C - and C 3- states and the diamagnetic C 2- state, of the 4Fe-4S cluster. Normally the cluster in any given protein undergoes one-electron redox changes between two of these states. In Chromatium HiPIP, the normal states are C - and C 2- ; the C 3- state is only achieved by partially unfolding the protein by treatments such as dimethylsulphoxide [6]. Bacterial ferredoxins are normally in the C 2- state and are reduced to the C 3- ~tate. However most preparations show a small EPR signal in the oxidized state, which has been attributed to the C - state by Sweeney et al. [7]. They showed that it could be enhanced by treatment with ferricyanide. We report here further investigations of the nature of the ferredoxin fractions from D. gigas. EPR spectroscopy is very suitable for observing the C - and C 3- states, which give rise to characteristic signals. The C - state has g-values slightly greater than the flee-electron value of 2.0023 [7], whereas the C 3- state gives an axial signal with gay around 1.96 [8]. The signals are only observed at low temperatures (10-50 K). By following the size of the EPR signals as a function of redox potential it is possible to determine the midpoint potentials of the C - / C 2- and C2-/C 3- tran3itions. All three oxidation states of the 4Fe-4S centre give rise to circular dichroism (CD) spectra in the visible and ultraviolet regions, the form of which is dependent on the type of protein [9]. This technique can therefore be used to observe the different forms of the proteins in all their oxidation states, including the diamagnetic C 2- state. MATERIALS AND METHODS The conditions for growth of the D. gigas cells and isolation of the three types of ferredoxin were as described by Bruschi et al. [1]. CD spectra were recorded on a FICA recording spectropolarimeter (SOFICA, St. Denis, France). EPR spcctra were recorded on a Varian E4 spectrometer (Varian Associates, Palo Alto, California, U.S.A.). Samples were cooled with liquid helium using a flow crystal (Oxford Instruments, Osney Mead, Oxford, U.K.). For quantitative EPR measurements, samples were prepared in quartz tubes matched for internal diameter (about 3.0 mm). Cu(II) EDTA was used as a standard. Spectra of the samples were lecorded under conditions of temperature and microwave power that did not saturate the signal intensity. The spectra were recorded digitally on a Nicolet 1020A digital oscilloscope (Nicolet Instrument Corp., Madison, Wisc. U.S.A.), interfaced to a HP 9830 calculator (Hewlett Packard Inc. Palo Alto, California, U.S.A.).

313 Integration was carried out numerically by the formula of Wyard [10] with a correction for slope in the baseline. A correction for the dependence of transition probability on g-value of the samples was made [11]. Oxidation-reduction titrations of the ferredoxin samples were carried out as previously described [12] in an apparatus similar to that of Dutton [13]. The ferredoxin, in 60 mM Tris HCI, pH 8.0, was poised at different potentials in the presence of the following mediators, all at 50/zM concentration; tetramethyl-p-phenylenediamine (E~ = ÷ 260 mV); 2,6-dichlorophenolindophenol (E~ = ÷ 217mV) 2,5dimethyllzenzoquinone (E~ = + 180 mV); phenazine methosulphate (E~ = 80 mV); methylene blue (E~ = 11 mV); indigotetrasulphonate (E~ = - - 4 6 mV); pyocyanin (E~ = -- 46 mV); 2-hydroxy-l,4-naphthoquinone (E~ = -- 145 mV); anthraquinone2-sulphonate (E~ = -- 225 mV); phenosafranine (E~ = -- 255 mV); benzylviologen (E~ = -- 345 mV); methyl viologen (E~ = -- 440 mV); and N,N'-dimethyl-3-methyl4,4'-bipyridyl (E~ = -- 617 mV). The potential was adjusted with small additions of dithionite or ferricyanide solution. After equilibration for I-5 min at a fixed potential a sample was taken and frozen for EPR measurements. RESULTS

EPR spectra The EPR spectra of the different forms of ferredoxins after reduction with dithionite (Fig. 1) are qualitatively similar to those observed in other four-iron ferredoxins such as Bacillus polymyxa [14] or Bacillus stearothermophilus [15]. The principal apparent g-values of FdI at 20 K are: gx = 1.92; gy = 1.94, gz = 2.07. The spectra of the three forms of D. gigas ferredoxins differed slightly in lineshape, particularly in the position of the central feature of the spectrum gy. Slight differences like these were observed between B. polymyxa ferredoxins I and II, which differ in their amino acid composition [14, 16]. In the case of D. gigas however, the amino acid composition is identical, so the differences are presumably due to differences in protein conformation or aggregation. The spectra were well resolved at temperatures below 50 K, and were readily saturated with microwave power at 10 K. This suggests a somewhat slower electron spin relaxation rate than in other reduced four-iron ferredoxins such as Bacillus stearothermophilus ferredoxin, which is only readily observed below 40 K [15]. The spectra of the oxidized proteins, as prepared, show EPR signals around g = 2, with a derivative peak at g = 2.02. These spectra are similar to those of other bacterial ferredoxins in the oxidized state [17] and have been attributed to the C state of the 4Fe-4S centre [7]. (We note that Dr. D. V. DerVartanian, of the University of Georgia, Athens, Ga., U.S.A. (personal communication) has also observed this signal in oxidized preparations of ferredoxin from D. gigas). Above 20 K the spectra of the oxidized proteins were narrow and almost structureless (Fig. 2a (i) and 2b (ii)), becoming broader with increasing temperature until at 40 K they were almost undetectable. At lower temperatures, structure appeared in the spectrum of FdI as seen in the shoulder to low field and the broadening to high field (Fig. 2a); FdI' was similar. The lineshape of FdlI, by contrast, remained constant down to 4.2 K with no sign of the appearance of additional structure. The three forms of ferredoxins differ substantially in the relative intensities

314 g -value 21

20

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(a)

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031

0.32

0.33 Magnetic

0.34

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0.35

0.36

field, T

Fig. 1. EPR spectra of the three forms of ferredoxins, reduced with dithionite: (a) FdI, 10 mg/ml; (b) FdI', 10 mg/ml; (c) FdlI, 15 mg/ml. Spectra were recorded at the following instrument settings: microwave power, 20roW; frequency 9.2 GHz; modulation amplitude 1 mT; temperature 18 K. The effective vertical expansion factors for the three spectra were 350, 560 and 3200, respectively.

of these signals. In the dithionite-reduced samples, the signal at g -- 1.94, corresponding to the C 3- state, is strong in FdI, less strong in FdI' and very small in FdlI even under optimum conditions of reduction. By contrast, in the samples as prepared, the g = 2.02 signal, corresponding to the C - state, is strong in FdlI, and weak in FdI: once again FdI' is intermediate. The integrated intensities of the signals are summarized in Table I. These results are consistent with the conclusions from N M R spectroscopy (Moura, J. J. G., Xavier, A. V., Bruschi, M. and Le Gall, J., Biochim. Biophys. Acta, in press) that FdI u n d e r g o e s oxidation-reduction between C 2- and C a- states, and FdlI between C - and C z- states. In FdI' substantial signals from either of the C - and C 3- states can be observed.

Oxidation-reduction properties On reduction with dithionite at pH 8.0, FdI gave the most rapid reduction, as shown by the development of the g = 1.94 signal. Maximal signal intensity was achieved after reduction with 5 m M dithionite f o r 2 min at 20 °C. For FdI', the reduction was slower, requiring 5-10 rain for maximum signal intensity. FdlI reacted sluggishly with dithionite, under the same conditions the g = 2.02 signal did not

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Fig. 2. EPR spectra of oxidized forms of (a) FdI, (b) FdlI. Spectra were recorded at temperatures: (i) 20 K, (ii) 12 K, (iii) 5.8 K. Other instrument settings were: microwave power 1 mW, frequency 9.12 GHz, modulation amplitude 0.5 mT. Gain settings were for (a) (i), 320; for (a) (ii)and (iii), 200; for (3) (i), 8; for (b) (ii), 5; for (b) (iii), 4. completely disappear for several minutes. Maximal development of the g = 1.94 signal required treatment with 20 mM dithionite for 10 min (Fig. 3a). High concentrations of dithionite caused a progressive loss of the EPR signals. Reduction of FdlI was more rapid in the presence of 0.1 mM methylviologen, and the maximal g = 1.94 signal intensity was then approximately twice as large (Fig. 3b). To attempt to further reduce the iron-sulphur centres in FdlI, it was treated with 80 % dimethylsulphoxide and reduced. Under these conditions, all proteins containing 4Fe-4S centres, including Chromatium HiPIP, tend to give the same type of spectrum [6, 18]. Fig. 3(c) shows the spectrum obtained; the intensity was considerably larger than that obtained by treatment with dithionite alone. The g-values, particularly gz, were shifted by the unfolding of the protein and are typical of 4Fe-4S proteins in dimethylsulphoxide [18]. The spectra of FdI and FdI' treated in the same way were very similar. A sample of FdlI was converted to the apo-form by treatment with trichloroacetic acid [19]. Electrophoresis of the protein in the presence of sodium dodecyl sulphate and urea indicated that the molecular weight of the apo-protein was close to that of the monomer, 6456 [2]. On reconstituting the protein with iron and sulphide in the presence of mercaptoethanol [19] a protein was formed which had no signal in the oxidized state and could readily be reduced by dithionite to the C 3- state, as indicated by the EPR spectrum (Fig. 3d). This reconstituted protein had the same mobility as FdI in 7.5 % polyacrylamide gel electrophoresis.

316 g -value. 2.1 r

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Fig. 3. Spectra of FdlI (a) reduced with 20 mM dithionite for 10 min. at 20 °C, gain 2000; (b) reduced with 5 mM dithionite ÷ 0.1 mM methyl viologen for 5 min, gain 2000; (c) treated with four volumes of dimethylsulphoxide and 5 mM dithionite [26]gain 2000; (d) reduced with 5 mM dithionite for 2 min., after conversion to the apo-form and reconstitution with iron and sulphide [19] gain 1000; the concentration of this sample is not directly comparable with the others. Other instrument settings were as for Fig. 1. Ferricyanide is an effective oxidizing agent, increasing the intensity of the g ---- 2.02 EPR signals due to the C - state in Clostridium pasteurianurn ferredoxin [7], but it was less effective in D. gigas ferredoxin. In FdI the size of the g = 2.02 signal could be increased by up to 2-fold. A fairly low ferricyanide concentration (about 0.1 mM) was found to be optimum. FdI' and FdlI showed little change. A consequence of the treatment with ferricyanide was a loss of the g = 1.94 signal that could be induced by dithionite; a similar effect has been noted with C. pasteurianum ferredoxin (Cammack, R., unpublished). Higher concentrations of ferricyanide caused destruction of the proteins. The destructive side-reactions of ferricyanide could be the reason why greater development of g = 2.02 signals was not observed (cf. 7). The midpoint potentials for the changes between the redox states of the three ferredoxins were determined by potentiometric titrations, monitored by EPR spectroscopy. The plots of the sizes of the principal spectral features, as a function of redox potential, are shown in Fig. 4. The midpoint potentials, derived by fitting to theo-

317

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Fig. 4. EPR signal intensities of (a) FdI; (b) F d I ' ; (c) F d I ] as a function of redox potential. For preparation of samples see Materials and Methods. II, g = 1.94 signal; 0 , g = 2.02 signal. In (b) open circles represent the intensity of the g = 2.02 signal after reduction to the lowest potential and

reoxidation with ferricyanide. Signal sizes were measured by the peak-to-dip size of the principal feature. EPR spectra were recorded under varying conditions of gain setting, and the signal intensities of the different proteins are not comparable with each other. For relative intensities see Table I. retical curves, are summarised in Table I. Because of the destructive effect of ferricyanide on the g = 1.94 signals, titrations were begun with the oxidized proteins and the potential of the samples was progressively reduced with small additions of dithionite. The reaction of FdII with the mediators was found to be slow, and an equilibration time of 5 min was allowed before taking each sample. It was found that after adjustment to the lowest potentials (which entails an excess of dithionite in the solution), the g = 2.02 signals seen on reoxidation were much smaller. This is illustrated for FdI' in Fig. 4(b), open circles. It appears therefore that just as treatment with ferricyanide decreased the size of the g -----1.94 signal that could be seen on reduction, treatment with dithionite decreased the size of the g = 2.02 signal that could be seen on reoxidation. From Table I it can be seen that the potentials for the C - / C 2- conversion in FdI, I' and II are all much more negative than the corresponding value of 350 mV for Chromatium high-potential iron-sulphur protein [21]. The value for FdII is particularly low. An even lower potential o f --420 mV has been reported for a similar reduction process of one of the 4Fe-4S centres in Azotobacter vinelandii ferredoxin

i [20].

318 The potentials for the C2-/C 3- conversion are also m o r e negative than those found in other 4Fe-4S ferredoxins, which lie between --280 and --380 mV [15, 22]. Circular dichroism Iron-sulphur proteins s h o w optically active transitions in the region 300-600 nm, corresponding to the absorption spectra [9]. Unlike the 2Fe-2S proteins, which (a) . . . .

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319 give relatively intense spectra with characteristic features [23], the 4Fe-4S proteins give rather weak spectra which vary with the type of protein; for example, the spectra of Clostridium ferredoxins [9] are quite unlike those of Chromatium HiPIP [4]. Although the CD spectra are not easy to interpret, they are characteristic of the particular oxidation state of a protein and are therefore useful in comparing the forms of D. gigas ferredoxin. Figs. 5(a) and (b) show CD spectra of FdI and FdlI respectively. The spectra of FdlI are generally more intense than FdI. There are features common to the spectra of both proteins, such as the negative band at 423 nm and the positive band around 580 nm, in the oxidized form of the proteins. In addition FdlI shows a positive band at 474 nm and a very intense negative band at 317 nm. The latter feature disappeared after reduction with dithionite and reoxidation with ferricyanide and the possibility cannot be excluded that it is due to a contaminant that was destroyed by the reduction and oxidation. However it is clear that the protein as a whole is modified by the dithionite/ferricyanide treatment, as seen in the decreased intensity of all of the features in Fig. 5(b) (iii). FdI was less severely affected by reduction and reoxidation (Fig. 5a) though the spectrum showed some enhancement of the feature at 474 nm after treatment with ferricyanide for about 1 h, indicating that the spectrum was becoming more like that of oxidized FdlI. In general the CD spectra of FdI and FdlI do not resemble those of other 4Fe-4S proteins, except in their intensity. This is surprising in view of the homology in amino acid sequence around the cysteine residues that bind the duster [2, 25]. Since the 4Fe-4S cluster has no inherent asymmetry the circular dichroism of the protein may be due either to distortion of the structure, or to the presence of asymmetric centres adjacent to the chromophore. Both of these are dependent on protein conformation, and might be expected to change with quaternary structure. In addition, EPR-silent forms of the protein produced by oxidative or reductive modifications, may also be contributing to the spectra. DISCUSSION In all three forms of D. gigas ferredoxin, FdI, I' and II, we observed EPR signals from the oxidized proteins and from the C 3- state on reduction with dithionite. However the sum of the integrated intensities of the signals of these two forms (Table I) never exceeded one unpaired electron per 4Fe-4S cluster. In other words, there is no evidence that a particular cluster can change reversibly between all three states C - , C 2- and C 3-. A more probable interpretation of the present evidence is that in some subunits of the proteins, the 4Fe-4S centres undergo the C - / C 2- transition as in Chromatium HiPIP. Other subunits have centres which undergo the C2-/C 3- transition. The latter subunits predominate in FdlI. FdI' may be considered as being composed of both types of subunit. The positions of four of the cysteine residues in the sequence of the basic unit of D. gigas ferredoxin of molecular weight 6456 are homologous with those that bind one of the clusters in Peptococcus aerogenes ferredoxin [24] and to the four cysteine positions in B. stearothermophilus ferredoxin [25]. The latter two proteins normally undergo oxidation-reduction between the C 2- and C 3- states. Therefore FdI is behaving as we would expect for such a protein and it follows that FdlI might be

320 TABLE I RELATIVE EPR SIGNAL INTENSITIES AND REDOX POTENTIALS FOR THE THREE FORMS OF D. GIGAS FERREDOXINS The g -- 2.02 signals in the oxidized (C-) state were measured in the proteins as prepared. The intensities of the g -- 1.94 signals in the reduced (C3-) state were measured in samples reduced with sufficient dithionite solution to give maximum signal intensities (see text). Spectra were recorded at 15 K at a microwave power of I roW. In each case, the intensity of the whole spectrum was integrated ; for the reduced signal, between 0.31 and 0.37 T, and for the oxidized signal, between 0.31 and 0.38 T. The method of integration was as described in the Materials and Methods section. Values are in electron spins per subunit molecular weight of 6456. Midpoint potentials were determined from the data of Fig. 4, and are expressed relative to the standard hydrogen electrode.

FdI FdI' FdII

Signal intensity (spins/6456 mol. wt.)

Midpoint potential (mV)

g = 2.02 g = 1.94

C-/C 2-

0.07 0.28 0.73

-- 50

--455

30 - 130

--430 -437

0.68 0.33 0.02

C2-/C 3-

p r o d u c e d f r o m F d I by some type o f modification which might be analogous to the treatment o f C. p a s t e u r i a n u m ferredoxin with ferricyanide [7]. This is supported by the observation that on forming the apo-protein o f F d l I and reconstituting, the p r o d u c t resembles F d I in that it can readily be reduced to the C 3- state. On this scheme, F d I ' represents an intermediate stage in this modification. On the basis o f the intensity o f the E P R spectra (Table I) it would be expected that the diamagnetic C 2- state predominates in the oxidized f o r m o f FdI and also in dithionite-reduced FdlI. However the C D spectra o f these forms (Figs. 5a (i) and 5b (ii)) show few similarities. This suggests that the C 2- state in F d I is not the same as the C ~- state o f FdlI, i.e. there is a modification o f the 4Fe-4S centre on formation o f FdlI. F d l I has a higher molecular weight than Fdl [1] indicating that its formation might be associated with a change f r o m a trimer to a tetramer form. However FdI' which behaves like F d l I in some ways, has the same molecular weight as FdI and a slightly different isoelectric point. It seems likely that the change involves both a change in quaternary structure and a modification at or near the iron-sulphur centre. The possibility that changes in quaternary structure o f the different oligomers can affect the relative stabilities o f the oxidation states o f the same monomeric unit, has been invoked to support the entatic state" theory [27] (Moura, J. J. G., Xavier, A. V., Bruschi, M. and Le Gall, J., Biochim. Biophys. Acta, in press). The nature o f the modification to the centre in F d l I is not clear, t h o u g h by analogy with C. p a s t e u r i a n u m ferredoxin it p r o b a b l y requires an oxidizing agent. It is possible that F d l I might be produced by the effects o f oxygen either before or after extraction; it should be remembered that the g ---- 2.02 signal is often seen in purified four- and eight-iron ferredoxins [7, 8]. However the observation [1] that F d l I is more efficient than F d I in an enzymic assay, coupling the electron transfer between hydrogenase and sulphite reduction, indicates that F d l I is probably not an artefact o f isolation, but has a physiological role in the organism.

321 ACKNOWLEDGEMENTS We are grateful to Miss C. P. Bargeron for skilled technical assistance. This work was s u p p o r t e d by grants from the U . K . Science Research C o u n c i l a n d the Royal Society, a n d the Institute de A l t a Cultura, Portugal. REFERENCES 1 Bruschi, M., Hatchikian, E. C., Le Gall, J., Moura, J. J. G. and Xavier, A. V. (1976) Biochim. Biophys. Acta 449, 275-284 2 Travis, J., Newman, D. J., Le Gall, J. and Peck, H. D. (1971) Biochem. Biophys. Res. Commun. 45, 452-458 3 Carter, C. W., Kraut, J., Freer, S. T., Alden, R. A., Sieker, L. C., Adman, E. and Jensen, L. H. (1972) Proc. Natl. Acad. Sci. U.S. 69, 3526-3529 4 Flatmark, T. and Dus, K. (1969) Biochim. Biophys. Acta 180, 377-387 5 Palmer, Go, Brintzinger, H., Estabrook, R. W. and Sands, R. H. (1967) in Magnetic Resonance m Biological Systems (Ehrenberg, A., Malmstr6m, B. G. and V~nng~.rd, T., eds.), pp. 159-171, Pergamon Press, Oxford 6 Cammack, R. (1973) Biochem. Biophys. Res. Commun. 54, 548-554 7 Sweeney, W. V., Bearden, A. J. and Rabinowitz, J. C. (1974) Biochem. Biophys. Res. Commun. 59, 188-194 80rme-Johnson, W. H. and Sands, R. H. (1973) in Iron-Sulfur Proteins (Lovenberg, W., ed.), Vol. 2, pp. 195-238, Academic Press, New York 9 Atherton, N. M., Garbett, K., Gillard, R. D., Mason, R., Mayhew, S. J., Peel, J. L. and Stangroom, J. E. (1966) Nature 212, 590-593 10 Wyard, S. J. (1965) J. Sci. Instrum. 42, 769-770 11 Aasa, R. and Vhnng~.rd, T. (1975) J. Mag. Res. 19, 308-315 12 Cammack, R., Barber, M. J. and Bray, R. C. (1976) Biochem. J. 157, 469-478 13 Dutton, P. L. (1971) Biochim. Biophys. Acta 226, 63-80 14 Stombaugh, N. A., Burris, R. H. and Orme-Johnson, W. H. (1973) J. Biol. Chem. 248, 7951-7956 15 Mullinger, R. N., Cammack, R., Rao, K. K., Hall, D. O., Dickson, D. P. E., Johnson, C. E., Rush, J. D. and Simopoulos, A. (1975) Biochem. J. 151, 75-83 16 Yoch, D. C. (1973) Arch. Biochem. Biophys. 158, 633-640 17 Palmer, G., Sands, R. H. and Mortenson, L. E. (1966) Biochem. Biophys. Res. Commun. 23, 357-362 18 Cammack, R. (1975) Biochem. Soc. Trans. 3,482-488 19 Hong, J. S. and Rabinowitz, J. C. (1967) Biochem. Biophys. Res. Commun. 29, 246-252 20 Sweeney, W. V., Rabinowitz, J. C. and Yoch, D. C. (1975) J. Biol. Chem. 250, 7842-7847 21 Dus, K., DeKlerk, H., Slatten, K. and Bartsch, R. G. (1967) Biochim. Biophys. Acta 140, 291-311 22 Zubieta, J. A., Mason, R. and Postgate, J. R. (1973) Biochem. J. 133, 851-854 23 Garbett, K., Gillard, R. D., Knowles, P. F. and Stangroom, J. E. (1967) Nature 215, 824-828 24 Adman, E. T., Sieker, L. C. and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987-3996 25 Hase, T., Ohmiya, N., Matsubara, H., Mullinger, R. N., Rao, K. K. and Hall, D. O. (1976) Biochem. J. 159, 55-63 26 Cammack, R. and Evans, M. C. W. (1975) Biochem. Biophys. Res. Commun. 67, 544-549 27 Vallee, B. L. and Williams, R. T. P. (1968) Proc. Natl. Acad. Sci. U.S. 59, 498-507