Communication Vol. 26 i , No. 21, Iscue of December 25, pp. 14620-14623, 1982 THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U S. A.

Evidence for Nickel and a Three- iron Center in the Hydrogenase of Desulfovibrio desulfuricans *

(Received for publication, August 18, 1982) Hans-Jorg KriigerS, Boi Hanh Huynh@Y, Peter 0. Ljungdahl$, Antonio V. Xavier(J**, Daniel V. DerVartanian,$$ Isabel Moura**, Harry D. Peck, Jr$& Miguel Teixeira**, Jose J. G. Moura**dO, and Jean LeGall$g@ From the + Department of Biochemistry, University of Georgia, Athens, Georgia 30602, the 7 Department of Physics, Emory University, Atlanta, Georgia 30322, and the ** Centro de Qucmica Estrutural das Uniuersidades de Lisboa, Znstituto Superior Tecnico, lo00 Lisbon, Portugal

Hydrogenase from Desulfovibrio desulfuricans (ATCC No. 27774) grown in unenriched and in enriched "Ni and 57Fe media has been purified to apparent ho- mogeneity. Two fractions of enzymes with hydrogenase activity were separated and were termed hydrogenase I and hydrogenase II. They were shown to have similar molecular weights (77,600 for hydrogenase I and 75,500 for hydrogenase 11), to be composed of two polypeptide chains, and to contain Ni and non-heme iron. Because of its higher specific activity (152 versus 97) hydrogen- ase II was selected for EPR and Miissbauer studies.

As isolated, hydrogenase I1 exhibits an "isotropic" EPR signal at g = 2.02 and a rhombic EPR signal at g = 2.3, 2.2, and 2.0. Isotopic substitution of "Ni proves that the rhombic signal is due to Ni. Combining the Mossbauer and EPR data, the isotropic g = 2.02 EPR signal was shown to originate from a 3Fe cluster which may have oxygenous or nitrogenous ligands. In addi- tion, the Mossbauer data also revealed two /4Fe-4Sl2+ clusters in each molecule of hydrogenase II. The EPR and Mossbauer data of hydrogenase I were found to be identical to those of hydrogenase 11, indicating that both enzymes have common metallic centers.

Hydrogenases have been purified to apparent homogeneity from a number of microbial species and found to exhibit a diversity (1) which is unexpected in view of the simplicity of the reaction involved in the activation of hydrogen. Generally, hydrogenases contain 4 to 12 atoms of non-heme iron/mole- cule. EPR studies suggest that the types of non-heme iron clusterfs) show considerable variability (1-3). Most recently, nickel has been shown to be required for the biosynthesis of hydrogenase (1, 4-7) and reported to be a structural compo-

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 Recipient of National Science Foundation Grant PCM-8108092, a Biomedical Research Support Grant, and a grant from the Emory Research Committee.

11 Recipient of National Institutes of Health Grant GM-25879. $$ Recipient of National Science Foundation Grant PCM-8111325

and a grant from the University of Georgia Research Foundation. 80 Recipient of National Science Foundation Grant PCM-8111325. 11 Recipient of a grant from the Junta Nacional de Investigacao

Cientifica e Technologica, and Gulbenkian Foundation, Portugal.

nent of the hydrogenases from Desulfovibrio gigas (2,8)' and Methanobacterium thermoautotrophicum (5). Both hydro- genases were demonstrated to contain 1 atom of nickel/mol- ecule; however, only the hydrogenase from D. gigas was demonstrated to contain EPR-active non-heme iron. In the oxidized form (ie. as isolated), both enzymes exhibit EPR signals with g values of 2.3, 2.2, and 2.0 (2, 8). Using isotopic substitution of "Ni, the EPR signal of Methanobacterium thermoautotrophicum hydrogenase was proven to have arisen from Ni (9). Based on the EPR spectrum originating from Ni(II1) in the membranes of M. bryantii (10, ll), the EPR signal from D. gigas hydrogenase was also proposed to reflect the presence of Ni(III), but in contrast to the hydrogenase fromM. thermoautotrophicum, reduced D. gigas hydrogenase exhibited Ni signals. Similar g = 2.3 EPR signals have been detected in a high molecular complex containing hydrogenase from M. thermoautotrophicum (12).

In this communication, we describe the purification and characterization of a hydrogenase from cells of Desulfouibrio desulfuricans grown in unenriched and enriched 61Ni and "Fe media. EPR signals with g values at 2.32, 2.21 and 2.01 have been unequivocally shown to be due to Ni, and Mossbauer studies indicate the presence of two [4Fe-4S] clusters plus a 3Fe cluster.

MATERIALS AND METHODS

Growth ofMicroorganisn and Preparation of Extract-D. desul- furicans (ATCC No. 27774) was grown in the medium described by Liu and Peck (13) containing nitrate rather than sulfate as a terminal electron acceptor thus avoiding precipitation of metal sulfides. For the growth of isotopically labeled cells, 40 mg of 6'Ni (enrichment 86.4%) and 400 mg of 57Fe (enrichment 95%) were first dissolved in H,SO, then in HC1, neutralized, and added to 400 ml of media. The "Ni was obtained from Oak Ridge National Laboratory and the "Fe from New England Nuclear. In a typical preparation of the crude extract, 350 g of cells were suspended in 1 liter of 10 m~ Tris-HC1, pH 7.6, and ruptured in a French press at 9,OOO ps i . under a N, atmos- phere. The extract was centrifuged at 19,OOO X g for 30 min and then at 180,000 X g for 75 min.

Assays-Hydrogenase activity was determined by the HZ evolution assay (14). Hydrogen was determined by means of a Varian 4600 gas chromotograph (2); iron by the method of Van De Bogart and Beinert (15); and sulfide by the method of Siege1 (16). Nickel was determined by plasma emission spectroscopy using the Jarrell-Ash model 750 Atomcomp. Protein was determined by t,he Bradford method (17) using bovine serum albumin as standard. The Bradford method was chosen because it gave values which were close to the ones obtained from calculations based on the extinction coefficient for D. gigas hydrogenase which has similar chromophore content.

Electrophoresis-Purity of the hydrogenase was established by polyacrylamide disc electrophoresis (18) as well as by sodium dodecyl sulfate-polyacrylamide electrophoresis (19).

Purification of Hydrogenase-For all purification procedures, the temperature was maintained at 5 "C and the pH of the buffers was 7.6 (measured at 5 "C for Tris-HCI). Precautions were taken against oxygen by flushing buffers with purified argon.

First DEAE-Bio-Gel Column-A DEAE-Bio-Gel column (8 X 22 cm) was prepared with 1100 ml of gel and successively washed with 300 ml of 1 M Tris-HCI, 300-400 ml of 10 mM Tris-HC1,300 ml of 0.5 M Tris-HC1 containing 10 mM Na&04, and 400 ml of 10 mM Tris- HCI. After the crude extract was loaded on the column, it was washed with 500 ml of 10 mM Tris-HC1 and the proteins were eluted with two Tris-HCI linear gradients (1 liter of 10 m~ Tris-HCI and 1 liter of 0.25 M Tris-HC1; 1 liter of 0.22 M Tris-HC1 and 1 liter of 0.4 M Tris-HCI).

' M. Teixeira, I. Moura, A. V. Xavier, D. V. DerVartanian, J. LeGall, H. D. Peck, Jr., B. H. Huynh, and J. J. G . Moura, submitted for publication.

14620

61Ni- and 57Fe-enriched Hydrogenase from D. desulfuricans 14621

The hydrogenase was collected in approximately 10-ml fractions between 2050 and 2800 ml.

First Hydroxylapatite Column-A hydroxylapatite column (4.5 X

with 300 ml of 1 M potassium phosphate buffer (KPB), 100 ml of 1 30.5 cm) was prepared with 485 ml of gel and washed successively

M Tris-HCI, 200 ml of 0.2 M Tris-HC1, 300 ml of 0.5 M Tris-HC1 containing 10 mM Na2SzO4, and 300 ml of 0.2 M Tris-HC1. After the hydrogenase-containing fractions from the first column were applied, the column was washed with 50 ml of 10 mM KPB and the proteins were eluted with two phosphate linear gradients (I liter of 10 mM KPB and 1 liter of 0.15 M KPB; 1 liter of 0.14 M KPB and 1 liter of 0.3 M KPB). The hydrogenase was eluted between 1600 and 2250 ml and concentrated to 90 ml in a Diaflow apparatus using a PM-30 mem- brane.

Second DEAE-Bio-Gel Column.-A DEAE-Bio-Gel column (6 X 21 cm) was prepared with 600 ml of gel and washed as previously described. The hydrogenase from the previous column was diluted with 150 ml of anaerobic water and applied to the column. After washing with 50 ml of 75 n m Tris-HC1, the column was developed with a Tris-HC1 linear gradient (1 liter of 75 mM Tris-HC1 and 1 liter of 0.25 M Tris-HC1). The hydrogenase eluted between 1310 and 1550 ml.

Second Hydroxylapatite Column-A hydroxylapatite column (4.5 X 21 cm) was prepared with 334 ml of gel and washed as described for the first hydroxylapatite column. After the hydrogenase from the previous step was absorbed on the column, it was washed with 50 ml of 10 mM KPB and the column was developed with a linear phosphate gradient (1 liter of 0.15 M KPB) at a rate of 40 ml/h. The hydrogenase was eluted in two bands: hydrogenase I, between 1270 and 1390 ml and hydrogenase 11, between 1710 and 1880 ml.

RESULTS AND DISCUSSION

The purification of the hydrogenases from D. desulfuricans is summarized in Table I. The overall recovery of hydrogenase activity was 13% and was divided about equally between two fractions containing hydrogenase activity, termed hydrogen- ase I and hydrogenase 11. Hydrogenase I1 was found to have higher specific activity than hydrogenase I. Hydrogenase I1 was demonstrated to have a molecular weight of 75,500 by ultracentrifugation and was judged to be homogeneous by polyacrylamide disc electrophoresis. On the basis of sodium dodecyl sulfate-polyacrylamide electrophoresis, both hydro- genases were demonstrated to be composed of two polypeptide chains. Hydrogenase I1 contains 10.9 atoms of iron, 11.4 atoms of acid-labile sulfur and 0.6 atom of Ni per molecule. Hydro- genase I has a molecular weight of 77,600 by ultracentrifuga- tion and appeared to be homogeneous by polyacrylamide disc electrophoresis. In addition to chromatographic behavior and specific activity, hydrogenase I differs from hydrogenase I1 in containing 7.8 atoms of iron, 6.8 atoms of acid-labile sulfur, and 0.6 atom of Ni per molecule. Since hydrogenase I1 ex- hibited the highest specific activity, it was subjected to de- tailed EPR and Mossbauer studies discussed below. The EPR

TABLE I Purification of hydroze

Fraction

Crude extract First DEAE-Bio-

First Hydroxylap-

Second DEAE-

Gel column

atite column

Bio-Gel col- umn

Second Hydroxyl- apatite col-

Hydrogenase I umn

Hydrogenase I1

. -

Volume

ml

.05 X 103 675

575

240

0.68 0.29

nases from D. des Protein

mg

22.7 X lo3 j.44 x 10

590

158

28 24

Total units

7, min” mmol

48.2 36.6

15.42

9.76

2.72 3.68

ul, furicans

3.2 5.69 1 76

I

and Mossbauer spectra of hydrogenase I are similar to those of hydrogenase 11, indicating similar prosthetic groups in both enzymes. The observed differences in activity and metal con- tent may suggest the presence of apoprotein in hydrogenase I.

The EPR spectrum of purified hydrogenase from D. desul- furicans exhibits an “isotropic” signal at g = 2.02 and a rhombic signal with g values at 2.32, 2.21, and 2.01. These values are slightly different but similar to those recorded for the purified hydrogenase from D. gigas (2). At low tempera- tures (2’ < 20 K), the EPR spectrum of hydrogenase I1 is dominated by an intense isotropic signal at g = 2.02. Addi- tional rhombic type signals are also observed. At temperatures higher than 77 K, the g = 2.02 signal becomes very broad and is no longer observable. Only the rhombic type signals remain. The high temperature EPR spectrum is relatively complex. Resonances are observed at g = 2.32, 2.21, 2.16, and 2.01 regions, indicating the existence of multiple EPR species. Due to its complexity, a definite assignment for the EPR reso- nances is difficult. However, comparing our results with the EPR data reported for purified hydrogenases from D. gigus (2), and from M. thermoautotrophicum ( 6 ) , as well as for a Ni(II1) species in oxidized membranes from M. bryantii, we have assigned the observed resonances at g = 2.32, 2.21, and 2.01 to one species and the additional resonance to other species.

In Fig. 1 the EPR spectra of hydrogenase I1 as isolated from cells of D. desulfuricans grown on both unenriched, and “Ni- and ”Fe-enriched media are compared at two temperatures, 11 and 90 K. Fig. 1A shows the spectra recorded at 90 K. The signals at 2.32 and 2.16 show significant broadening for the

Ni-enriched hydrogenase when compared to the naturally occurring purified hydrogenase. In the g = 2.01 region, hyper- fine structure is observed for the ‘lNi-enriched protein. Lan- caster (11) reported four distinct hyperfine lines in this region in the M. Bryantii “Ni-enriched system due to the nuclear spin (I = ‘Ti) of “Ni. It is interesting to note that the separation between the two outermost lines for the “Ni-enriched hydro- genase is 79 G while that measured from Lancaster’s data is 80 G. Thus, the hyperfine patterns for ”Ni show both broad- ening and partial resolution of hyperfine lines indicating un- equivocally that the rhombic signal observed in the oxidized state of hydrogenase is due to nickel.

Since the inner two hyperfine lines are not well resolved, and the enzyme is also enriched in 57Fe, the observed hyperfine pattern could be due to the nuclear spin ( I = %) of 57Fe. However, a hyperfine splitting of 80 G observed in an EPR spectrum for an electronic spin ?h system corresponds to an internal field of 800 kG at the “Fe nucleus. The following Mossbauer data (Table 11) completely rule out this possibility.

Fig. 1B shows the EPR spectrum of purified hydrogenase recorded a t 11 K in the g = 2.02 region. Hyperfine broadening due to the 57Fe nuclear spin is very definitely observed in the 57Fe-enriched hydrogenase. The half-width signal of the ”Fe- hydrogenase signal is 21 G while that of the “Fe-enriched hydrogenase signal is 32 G, indicating a broadening of 11 G. There also appears to be hyperfine structure in the “Fe- enriched hydrogenase spectrum with an apparent splitting constant of approximately 13 G. These values of splitting constants are within the range of those found in iron-sulfur clusters, and agree very well with the magnetic hyperfine coupling constants obtained for a paramagnetic component observed in the 57Fe Mossbauer spectra (Table 11).

Upon reduction by hydrogenase, the isotropic g = 2.02 signal and the rhombic Ni signal disappear and are replaced by an intense “g = 1.94 type reduced [4Fe-4S]” signal and additional weak signals presumably due to nickel. The new

61

14622

g= 2f2

6'Ni- and "Fe-enriched Hydr

*f6 2.01 1

n

A

g = 2.02 1

B

FIG. 1. The effects of isotopic substitutions on the EPR spec- trum of the hydrogenase from D. desulfuricans. A, purified hydrogenase 11 (70 PM in protein in 100 mM potassium phosphate buffer, pH 7.6). Lower trace, purified hydrogenase enriched in "'Ni. EPR conditions: microwave power, 10 milliwatts (in each instance was performed under nonsaturating conditions); microwave fre- quency, 9.154 GHz; modulation amplitude, 10 G; temperature, 99 K; scanning rate, 400 G/min; time constant, 0.1 s. The EPR traces in A are the average of 9 scans. Gain (upper trace) = 6.25 X 10'. Gain (lower trace) = 3.2 X 10". B, purified hydrogenase (as in Fig. IA) measured under the following EPR conditions: microwave power, 20 microwatts; microwave frequency, 9.155 GHz; modulation amplitude, 5 G; temperature, 12 K; scanning rate, 50 G/min; time constant, 0.1 s. Gain (natural abundant "'Fe hydrogenase) = 2.5 X 10' and gain ("Fe- enriched hydrogenase) = 3.2 X 10".

TABLE I1 Mossbauer parameters for the paramagnetic iron center in purified

hydrogenase from D. desulfuricans The quadrupole splittings AEQ and isomer shifts 6 (relative to iron

metal at room temperature) are values obtained at 85 K. 1) is the asymmetry parameter. g.p. is the nuclear magnetic moment. With the limited resolution, the A-tensor is assumed to be isotropic for site 2 and is undetermined for site 3. The values in parentheses are equivalent gauss at the electron. These values are consistent with the hvoerfine seDarations observed in the isotrooic e = 2.02 sienal.

nickel signal has maximum g value at 2.28 and its intensity is about one-third of the g = 2.32 resonance. These observations will be the subject of a future communication.

Fig. 2 shows Mossbauer spectra of "Fe-enriched hydrogen- ase I1 from D. desulfuricans. The data are recorded at 4.2 K with a magnetic field of 500 G applied parallel (Fig. 2 A ) and perpendicular (Fig. 2B) to the y-radiation. The spectrum

-ogenase from D. desulfuricans

consists of at least two subspectral components: an intense quadrupole doublet at the center and a paramagnetic com- ponent extended from -2 to +3 mm/s.

The doublet accounts for approximately 70-80% of the total iron absorption. The observed quadrupole splitting (AEQ = 1.17 mm/s), the isomer shift (6 = 0.43 mm/s), and the general shape of the doublet are typical of a [4Fe-4SI2' cluster (20, 21). These observations, together with the iron quantitation, indicate that hydrogenase 11, as prepared, contains two [4Fe- 4S] clusters in the 2+ oxidation state.

A close examination of Fig. 2, A and B, reveals that the absorption pattern of the magnetic component is field direc- tion-dependent. To study the magnetic component in more detail, spectrum 2B is subtracted from spectrum 2A, and the difference spectrum is shown in Fig. 2C. The absorption of the intense doublet is cancelled in the difference spectrum because the [4Fe-4SI2+ cluster is diamagnetic and its spectrum will not be affected by changing direction of the applied field. Two important observations are evident in Fig. 2C. First, the magnetic component is strongly field direction-dependent, a phenomenon which suggests that an intense EPR signal should be associated with the magnetic component. This EPR signal is identified as the isotropic g = 2.02 signal. Second, at least two pairs of Am = 0 nuclear transitions are observed (indicated by the brackets). Since a single iron site can only have one pair of Am = 0 transitions, the above observation demonstrates that at least two iron sites are associated with the magnetic components.

We have analyzed the spectra of the two discernible mag- netic iron sites using an S = % spin Hamiltonian,

I

" I - z l

1

I I I 1 I I I I I 1

VELOCI TY (mm/s 1 -4 -2 0 2 4

FIG. 2. Mossbauer spectra of "Fe-enriched hydrogenase I1 from D. desulfuricans. The spectra are recorded at 4.2 K with a magnetic field of 500 G applied parallel (A ) and perpendicular ( B ) to the y-radiation. A difference spectrum of spectra A and B is shown in C (see text). The brackets indicate the two pairs of Am = 0 nuclear transitions. The solid lines are theoretical spectra for the two dis- cernible iron sites of the g = 2.02 paramagnetic center. The parameters used for the theoretical computations are listed in Table 11.

61 Ni- and “Fe-enriched Hydrogenase from D. desulfuricans 14623

H = & H . s + s . A . I +- ” - ” eQVzz [3Z2 - 1(1

12

+ 1) + ?(I2 - I?)] - g,p,ti.i (1)

All symbols in Equation 1 have their conventional meanings (22). The solid lines in Fig. 2 are theoretical simulations. The parameters used for calculations are listed in Table 11. By comparing the absorption intensities of the peaks at -3 and -2 mm/s with those of the simulated spectra, the two iron sites are estimated to contribute 16-20% of the total iron absorption.

The EPR studies showed that at temperatures higher than 77 K, the g = 2.02 signal disappeared, indicating fast electronic relaxation rate. Consistent with this EPR observation, the Mossbauer magnetic component collapses into a sharp quad- rupole doublet at 85 K. The measured parameters (AEQ = 0.63 mm/s and 6 = 0.36 mm/s) are typical for high spin ferric ion (S = Yz). However, two S = 5/2 irons cannot be coupled to form a total spin of %. A third half-integral spin is required. Also the absorption intensity of the collapsed quadrupole doublet was found to be 25-30% of the total absorption, which is about % of that of the two discernible magnetic iron sites. We therefore conclude that the g = 2.02 center must contain a third iron site which at low temperature spectrum is masked by the intense central doublet.

Three-iron clusters have recently been found in various proteins (23-25). The physical properties of the g = 2.02 center in hydrogenase are very similar to those of a 3Fe cluster found in Azotobacter vinelandii ferredoxin (23). However, all the oxidized 3Fe clusters reported so far have an isomer shift less than 0.3 mm/s, a typical value for high spin ferric ion in a tetrahedral sulfur environment. In D. desulfuricans hydro- genase a relatively larger isomer shift, 0.36 mm/s, was found, suggesting that the iron coordination of this trinuclear iron cluster may contain nitrogenous or oxygenous ligands.

The hydrogenases isolated from D. gigas (2, 26, 27), D. desulfuricans, and the hydrogenase of D. vulgaris (28, 29) exhibit significant differences in their molecular organization and specific activity. However, evidence collected from chem- ical analyses, EPR, and Mossbauer studies indicate that all three enzymes contain some common prosthetic groups. All three hydrogenases were reported to contain approximately 11-12 non-heme irons and comparable amounts of acid-labile sulfurs per molecule. Approximately 1 atom of nickel was found in hydrogenase from D. gigas (2) and from D. desulfur- icans but there has been no report of Ni in hydrogenase from D. vulgaris. The EPR and Mossbauer spectra of isolated hydrogenases from D. gigas and from D. desulfuricans are practically the same, suggesting similar organization for the iron and sulfur atoms in the enzymes f i x . two [4Fe-4S] clusters and one 3Fe cluster per molecule). As isolated, the hydrogenase from D. vulgaris exhibits a g = 2.02 EPR signal which is lost upon reduction and replaced by a low intensity g = 1.94 type signal (3). These signals are consistent, respec- tively, with oxidized 3Fe cluster and reduced ferredoxin type [4Fe-4S] cluster.

Recently, it has been shown that [4Fe-4S] clusters can be converted into 3Fe clusters by ferricyanide oxidation (30-32). The activation of beef heart aconitase was shown to involve the transformation of 3Fe clusters into [4Fe-4S] clusters (33). Also, the conversion of 3Fe to [4Fe-4S] clusters was achieved in D. gigas ferredoxin system by both incubation with iron in reducing conditions and reconstitution procedures (30). These findings raised the question whether the 3Fe clusters have a physiological role or are the result of oxidative damage of [4Fe-4S] clusters during protein purification. The D. desul- furicans hydrogenase was purified under anaerobic proce-

L.

3.

4.

6. 5.

7. 8.

9.

10.

12. 11 .

13. 14. 15. 16. 17. 18.

20. 19.

21.

22.

23.

24.

25.

26. 27.

28.

29.

30.

dures, and it is therefore rather unlikely that the presence of 3Fe clusters in this enzyme is due to oxidation damage of [4Fe-4S] clusters.

Although nickel is not yet established as an essential com- ponent of all hydrogenases, it is found in hydrogenases purified in various species. Evidence gathered so far indicates that the nickel center is sensitive to the redox state of the enzyme (2), and it can undergo a one-electron reduction from Ni(II1) to Ni(I1) with pH-dependent midpoint redox potential (2, 8). These results suggest that nickel must play an important role in the activation of hydrogen in these hydrogenases.

Acknowledgments-We wish to thank Susan K. Holmes for the excellent technical assistance with the computer-assisted EPR spec- troscopy, Ned Rich and Dr. John Wampler for interfacing and pro- gramming the Varian E-109 spectrometer with the Hewlett-Packard 85 microcomputer for data acquisition, and the staff of the University of Georgia Fermentation Plant for the growth of D. desulfuricans.

REFERENCES

1. Adams, M. W. W., Mortenson, L. E., and Chen, (1981) Biochim. Biophys. Acta 594, 105-176

LeGall, J., Ljungdahl, P. O., Moura, I., Peck, H. D., Jr., Xavier, A. V., Moura, J. J. G., Teixeira, M., Huynh, B. H., and DerVartanian, D. V.

Van Dijk, C., Grande, H. J., and Veeger, C. (1982) in Photochemical, (1982) Biochem. Biophys. Res. Commun. 106,610-616

Photoelectrochemical and Photobiological Processes (Hall, D. O., and Palz, W., eds) series D, Vol. 9, pp. 190-194, D. Reidel Publishing Co.,

Friedrich, B., Heine, E., Finck, A,, and Friedrich, C. G. (1981) J. Bacteriol. Dordrecht, The Netherlands

Graf, E.-G., and Thauer, R. K. (1981) FEBS Lett. 136, 165-169 145, 1144-1149

Tzkakowa, S., and Wall, J. D. (1981) FEBS Lett. 12,359-363 Partridge, C. D. P., and Yates. (1982) Biochem. J. 204, 339-344 Cammack, R., Patil, D., Aguirre, R., and Hatchikian, E. C. (1982) FEBS

Albracht, S. P. J., Graf, E.-G., and Thauer, R. K. (1982) FEBS Lett. 140,

Lancaster, R. J., Jr. (1980) FEBS Lett. 115, 285-288 Lancaster, R. J., Jr. (1982) Science (Wash. D. C.) 216, 1324-1325 Jacobson, F. S., Daniel, L., Fox, J. A., Walsh, C. T., and Orme-Johnson, W.

Liu, M. C.. and Peck, H. D., Jr. (1981) J. Biol. Chem. 256, 13159-13164 Peck, H. D., Jr., and Gest, H. (1956) J. Bacteriol. 71, 70-80 Van De Bogart, M., and Beinert, H. (1967) Anal. Biochem. 20,325-334 Siege], L. M. (1965) Anal. Biochem. 11, 126-132 Bradford, M. M. (1976) Anal. Biochem. 72,248-254 Brewer, J. M., and Ashworth, R. B. (1969) J. Chem. Educ. 46, 41-45 Weber, K., and Osbom, M. (1969) J. B i d . Chem. 244,4406-4412 Middleton, P., Dickson, D. P. E., Johnson, C. E., and Rush, J. D. (1980)

Middleton, P., Dickson, D. P. E., Johnson, C. E., and Rush, J. D. (1978) Eur. J. Biochem. 104,289-296

Munck, E., and Huynh, B. H. (1979) in ESR and NMR of Paramagnetic Eur. J . Biochem. 88, 135-141

Species in Biological and Related Systems (Bertini, I., ed) NATO Advanced Study Institutes Series C, Vol. 52, pp. 275-288 D. Reidel

Emptage, M. H., Kent, T. A., Huynh, B. H., Rawlings, J., Orme-Johnson, Publishing Company, Dordrecht, The Netherlands

W. H., and Munck, E. (1980) J. Biol. Chem. 255, 1793-1796 Huynh, B. H., Moura, J. J. G., Moura, I., Kent, T. A,, LeGall, J., Xavier, A.

Kent, T. A., Dreyer, J.-L., Kennedy, M. C., Huynh, B. H., Emptage, M. H., V., and Munck, E. (1980) J. Biol. Chem. 255,3242-3244

Beinert, H., and Munck, E. (1982) Proc. Natl. Acad. Sci. U. S.A. 79, 1096-1100

Bell, G. R., LeGall, d. , and Peck, H. D., Jr. (1974) J. Bacteriol. 120,994-997 Hatchikian, E. C., Bruschi, M., and LeGall, d . (1978) Biochem. Biophys.

Van der Westen, H. M., Mayhew, S. G., and Veeger, C. (1978) FEBS Lett Res. Commun. 82, 451-461

Mayhew, S. G., van Dijk, C., and Van der Westen, H. M. (1978) in 86, 122-124

Hydrogenases: Their Catalytic Actiuity, Structure and Function, (Schle- gel, H. D., and Schneider, K., eds) pp. 125-140, E. Goltze K. C., Gottingen

Moura, J. J. G., Moura, I., Kent, T. A., Lipscomb, J. D., Huynh, B. H., LeGall, J., Xavier, A. V., and Munck, E. (1982) J. B L O ~ . Chem. 257, 6259- 6267

Lett. 142,289-292

311-313

H. (1982) J. Biol. Chem. 257,3385-3388

31. Thomson, A. J., Robinson, A. E., Johnson, M. K., Cammack, R., Rao, K. K., and Hall, D. 0. (1981) Biochim. Biophys. Acta 637,423-432

32. Johnson, M. K., Spiro, T. G., and Mortenson. L. E. (1982) J. Biol. Chem. 257,2447-2452

33. Kent, T. A., Dreyer, J.-L., Kennedy, M. C., Huynh, B. H., Emptage, M. H., Beinert, H., and Munck, E. (1982) Proc. Natl. Acad. Sci. c! S.A. 79, 1096-1100