3576
J. Am. Chem. SOC.1993,115, 3576-3585
An X-ray Absorption Spectroscopic Study of Nickel Redox Chemistry in Hydrogenase Csaba Bagyinka,t*tJoyce P. Whitehead,+and Michael J. Maroney'*tvl Contribution from the Department of Chemistry and Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003 Received June 29, 1992
Abstract: The results of X-ray absorption spectroscopic studies of Thiocapsa roseopersicina hydrogenase poised in three forms exhibiting EPR signals due to the Ni center (A, B, and C) and two states that are epr silent with respect to the Ni center are reported. These spectra are used to examine the structural changes that occur during the reduction of the enzyme. Analyses of Ni K-edge spectra reveal the presence of weak features at ca. 8332 eV in the spectra obtained from forms A and B and the silent intermediate (SI) that are assigned to 1s 3d transitions. The lack of a significant pre-edge peak in the active form of the enzyme and low peak areas in other forms, coupled with the absence of edge features associated with planar four-coordinate Ni complexes, indicate that the Ni site in all states of the enzyme is five- or six-coordinate. N o observable shift in edge energy occurs upon reduction of the enzyme to any level. This demonstrates that no significant change in the electron density of the Ni site occurs during reduction. Analyses of the EXAFS spectra obtained from scattering atoms in the first coordination sphere of Ni in all five states of the enzyme that are defined by Ni EPR signals (or lack thereof) are consistent with a Ni site composed of 3 f 1 N(0)-donors a t 2.00 f 0.06 A and 2 f 1 S-donors at 2.23 f 0.03 A. These results are discussed in light of various models for the structure and function of the Ni site in the enzyme. N o evidence to support a redox role for Ni in hydrogenase is found in the XAS data.
-
pterin dehydrogenase from Methanobacterium thermoautoHydrogenases (Hzases) are a diverse group of enzymes found in both prokaryotes and eukaryotes that catalyze the reversible trophicum that possesses H2ase activity but does not contain two-electron oxidation of molecular hydrogen. 1-3 Thus, H2ases Fe,14 all H2ases contain Fe,S clusters. One class, the Fe-only enzymes, contains only Fe and sulfide.] Since the discovery of may function to provide reducing equivalents for energy proNi as a biological component of Methanobacterium bryantii in duction via the uptake and oxidation of H2 or they may reduce 198015 and the subsequent identification of the Ni-containing Hf in the production of H2. Hydrogen oxidation (uptake) is component as a H2ase,16 many examples of H2ases that require generally coupled with phosphorylation and ultimately to the a single Ni atom as well as Fe,S clusters have been characterized. reduction of inorganic substrates such as Sod2-(Desulfovibrio Hzases belonging to the Fe,Ni class are generally associated with specie^),^ C02 (Methanobacterium specie^),^ N03- (e.g. Paraoxidation in vivo and are by far the most common and coccus denitri'can~),~.~ or 0 2 (Alcaligenes and N o ~ a r d i a ) . ~ . ~ .hydrogen ~ widely distributed form of the enzyme; examples are known from H2ases may also play a role in cycling hydrogen produced in fermentative,17 S 0 4 - r e d u ~ i n g , ~mq e~t~h a n ~ g e n i c photosyn,~ other systems (e.g. nitrogenase) or in generating a proton the ti^,^^,^^ facultative,20and aerobic bacteria.21,22 Among the gradient.1° Hzases containing Ni are a class of enzymes that contain Se as Hydrogenases have been grouped into three classes, based on well-the Fe,Ni,Se H 2 a ~ e s . l ~The Se is generally present as a the inorganic content of the enzymes, that are immunologically single selenocysteine residue, most notably in D . baculatus,where and biochemically distinct.]]-I3 With the apparent exception of it has been shown to be encoded by an internal TGA codon,23and a recently purified enzyme, W,IVO-methylenetetrahydromethano constitutes a conservative replacement for a cysteine residue in Department of Chemistry. enzymes lacking selen~cysteine.~~ Spectroscopic studies have t Current Address: Biological Research Center of the Hungarian Academy shown this selenocysteine to be one of the Ni l i g a n d ~ . ~ ~ . ~ 5 of Sciences, H-6701, Szeged, Hungary. When Ni is present in the enzyme, it is often detected by an f Program in Molecular and Cellular Biology. (1) Adams, M. W. W. Biochim. Biophys. Acta 1990, 1020, 11545. unusual rhombic S = ' / 2 EPR signal ( g = 2.4-2.0) that has been (2) Cammack, R.; Hall, D. 0.;Rao, K. K. In Microbial Gas Metabolism: Mechanistic, Metabolic and Biotechnical Aspects; Poole, K. K., Dow, C.
Eds.; Academic Press: London, 1985; Chapter 4. (3) Przybyla, A. E.; Robbins, J.; Menon, N.; Peck, H. D. FEMSMicrobiol. Rev. 1992, 88, 109-35. (4) Hatchikian, E. C.; Fernandez, V. M.; Cammack, R. FEMS Symp. 1990,54, 53-73. (5) Bastian, N. R.; Wink, D. A.; Wackett, L. P.; Livingston, D. J.; Jordan, L. M.; Fox, J.; Orme-Johnson, W. H.; Walsh, C. T. In The Bioinorganic Chemistry of Nickel; Lancaster, J. R., Jr., Ed.; VCH: New York, 1988; pp 221-47. (6) Knuttel, K.; Schneider, K.; Schlegel, H. G.; Muller, A. Eur. J . Biochem. 1989, 179, 101-8. (7) Sim, E.; Vignais, P. M.; Biochimie 1978, 60, 307-14. (8) Schink, B.; Schlegel, H. G. Biochemie 1978, 60, 297-305. (9) Doyle, C. M.; Arp, D. J. J . Bacteriol. 1987, 169, 4463-8. (10) Vignais, P. M.; Colbeau, A,; Willison, J. C.; Jouanneau, Y . Adu. Microb. Physiol. 1985, 26, 155-234. (11) Kovacs, K. L.; Seefeldt, L. C.; Tigyi, G.; Doyle, C. M.; Mortensen, L. E.; Arp, D. J. J . Bacteriol. 1989, 171, 430-5. (12) Fauque, G.;Teixeira, M.; Moura, I.; Lespinat, P. A,; Xavier, A. V.; Der, V. D.; Peck, H. J.; Le, G. J.; Moura, J. G. Eur. J . Biochem. 1984, 142, 21-8.
(13) Lorenz, B.; Schneider, K.; Kratzin, H.; Schlegel, H. G. Biochim. Biophys. Acta 1989, 995, 1-9.
(14) Zirngibl, C.; Hedderich, R.; Thauer, R. K. FEBS Lett. 1990, 261, 112-16. (15) Lancaster, J. R., Jr. FEBS Lett. 1980, 115, 285-88. (16) Albracht, S. P.; Graf, E. G.; Thauer, R. K. FEBS Lett. 1982, 140, 311-3. (17) Bryant, F. 0.;Adams, M. W. J . B i d . Chem. 1989, 264, 5070-9. (18) Fauque, G.; Peck, H. D., Jr.; Moura, J. J. G.;Huynh, B. H.; Berlier, Y . ;DerVartanian, D. V.; Teixeira, M.; Przybyla, A. E.; Lespinat, P. A.; Moura, I.; LeGall, J. FEMS Microbiol. Reu. 1988, 54, 299-344. (19) Gogotov, I. N. Biochemie 1986, 68, 181-7. (20) Ballantine, S. P; Boxer, D. H. J . Bacteriol. 1985, 163, 454-9. (21) Cammack, R.; Fernandez, V. M.; Schneider, K. In The Bioinorganic Chemistry of Nickel; Lancaster, J. R., Jr., Ed.; VCH: New York, 1988; Chapter 8. (22) Seefeldt, L. C.; Arp, D. J. Biochimie 1986, 68, 25-34. (23) Voordouw, G.; Menon, N. K.; LeGall, J.; Choi, E. S.; Peck, H. J.; Przybyla, A. E. J . Bacreriol. 1989, 171, 289&9. (24) Eidsness, M. K.; Scott, R. A,; Prickril, B. C.; DerVartanian, D. V.; Legall, J.; Moura, I.; Moura, J. J.; Peck, H. J. Proc. Natl. Acad. Sci. U.S.A. 1989.86, 147-51.
0002-7863/93/1515-3576$04.00/00 1993 American Chemical Society
J. Am. Chem. SOC.,Vol. 115, No. 9, 1993 3577
Study of Nickel Redox Chemistry in Hydrogenase Table I. Amino Acid Analysis of Thiocapsa roseopersicina Hydrogenase amino acid
X f fl
Kovacs et aL3I
amino acid
Asp Glu Ser Gly His Arg Thr Ala Pro
103.0f 3.1 80.5 f 5.4 43.2 f 2.8 84.0 f 3.3 18.5 f 2.1 57.2 f 1.5 50.8 f 0.4 105.2 f 1.5 48.2 f 4.8
136 48 68 136 20 25 44 124 50
Tyr Val Met Ile Leu Phe Lys Cys Trp
X f fl
Kovacs et aL3'
30.5 f 1.7 66.5 f 1.5 21.3 f 0.9 49.5 f 1.8 87.8 f 2.9 32.7 f 1.8 32.0 f 4.1 12.5 f 0.5
4 60 4 32 54 18 19 4
a Based on the analysis of four samples except for cysteine/cystine, which is based on two samples. Analysis was not performed for Trp.
assigned to formally Ni(II1) or Ni(1) center^.^^,^' Distinct EPR signals are observed for the oxidized and catalytically inactive forms of the enzyme (forms A and B), as well as in a reduced and active form (form C). These EPR signals have provided a principal spectroscopic probe of the Ni site and have been used to demonstrate the redox activity of the site.21,26,28Under H2 atmosphere, the signals associated with the oxidized forms disappear, yielding a redox state of the enzyme that is EPR silent at 77 K (silent intermediate, SI). Further exposure to H2 causes the signal associated with the active form of the enzyme to appear. Extensive incubation of the enzyme under H2 leads to the formation of the fully reduced form (R) of the enzyme, which is also EPR silent. Various schemes employing formal Ni oxidation states IV-0 have been used to account for the appearance and disappearance of EPR signals associated with the Ni site.5~21~26~28,29 Using samples of Thiocapsa roseopersicina H2ase, a typical Fe,Ni enzyme,19x21*3k32 poised with respect to the Ni EPR signals, we report here the results of a Ni K-edge X-ray absorption spectroscopic study of the structure of the Ni site in each form of the enzyme. This study reveals structures for the Ni site that are remarkably insensitiue to the redox state of the enzyme and are therefore inconsistent with Ni-centered redox chemistry.
Results Enzymology. Samples of Thiocapsa roseopersicina H2ase were analyzed for amino acid content in order to obtain an accurate measure of the protein concentration of samples used in the XAS study. The results of these analyses are summarized in Table I. Two previous analyses of the amino acid content of this enzyme have appeared in the 1iterature3OJ2and are not in agreement with each other regarding the amino acid content of the enzyme. Our results do not agree with either of the previously published determinations. Nonetheless, the analyses reported here are reproducible and provide an estimate of the molecular weight of the protein of 98.0 kDa, which is in agreement with published values.32 The electronic absorption spectra of the samples that were analyzed for amino acid content were used to obtain extinction coefficients for the enzyme a t 220 nm (895 000 cm-l (25) He, S. H.; Teixeira, M.; LeGall, J.; Patil, D. S.;Moura, I.; Moura, J. J.; DerVartanian, D. V.; Huynh, B. H.; Peck, H. J. J . Biol. Chem. 1989, 264, 2678-82. (26) Moura, J. J. G.;Teixeira, M.;Moura, I.; LeGall, J. In The Bioinorganic Chemistry ofNickel; Lancaster, J. R., Jr., Ed.; VCH: New York, 1988; pp 19 1-226. (27) Moura, J. J. G.; Teixeira, M.; Xavier, A. V.; Moura, I.; LeGall, J. J . Mol. Catal. 1984, 23, 303. (28) van der Zwaan, J. W.; Albracht, S. P.; Fontijn, R. D.; Slater, E. C. FEES Lett. 1985, 179, 271-7. (29) Coremans, J. M. C. C.; VanderZwaan, J. W.; Albracht, S. P. J . Biochim. Biophys. Acta 1989, 997, 256-61. (30) Gogotov, I. N.; Zorin, N. A.; Serebriakova, L. T.; Kondratieva, E. N . Biochim. Biophys. Acta 1978, 523, 335-43. (31) Kovacs, K. L.; Bagyinka, C. FEMS Microbiol. Rev. 1990, 87, 40711.
(32) Kovacs, K. L.;Tigyi, G.;Thanh, L. T.; Lakatos, S.;Kiss, Z.;Bagyinka, C. J . Biol. Chem. 1991, 266, 947-51.
SI
+
1
t
g=2.01 g=2 9
Form C
R
I
2800
I
I
I
3000 3200 3400 Magnetic Field (Gauss)
I
3600
Figure 1. Nickel EPR spectra of Thiocapsa roseopersicina hydrogenase X A S samples prior to exposure to synchrotron radiation. The spectra were recorded at 77 K, with microwave power of 20 mW, at a frequency of 9.62 GHz, and a modulation amplitude of 4 G.
M-I) and 280 nm (106 000 cm-1 M-I). Using the absorbance at 220 nm and BSA as a standard to calculate the protein concentration of the samples leads to values that are 84.7% of the total protein amount determined by the amino acid analysis. A correction factor of 1.18 was therefore applied to the apparent concentration of H2ase determined using spectrophotometric measurements and a standard BSA calibration curve.33 Metal analyses were performed on a portion of the samples analyzed for amino acid content and on samples where the concentration of the protein was determined spectrophotometrically. These analyses reveal that the enzyme contains 1.0 f 0.1 Ni atoms and 8.0 f 1.OFe atoms per molecule, in agreement with previously determined Ni:Fe rati0s.~*J3 Samples of T . roseopersicina H2ase were poised with respect to the N i EPR signal at 77K using the procedures described in the Experimental section. These samples were frozen in polycarbonate holders and the EPR spectra of the samples before exposure to synchrotron radiation were measured. Representative spectra are shown in Figure 1. Redox levels that have Ni EPR signals, where we can ascertain that the majority of the ~~
(33) Maroney, M. J.; Colpas, G. J.; Bagyinka, C.;Baidya, N.;Mascharak, P. K. J . Am. Chem. SOC.1991, 113, 3962-72. (The estimate of the redox poise of the form C sample used in this previous study has been corrected from 95% to 80% based on the better determination of the protein content of the sample available from amino acid analysis.)
3578 J. Am. Chem. SOC.,Vol. 115, No. 9, 1993 Table 11. EPR and Ni K-Edge Data enzyme redox state form A( 1) form A(2) form A(3)
Ni K-edge energy *0.2 (eV) 8339.6 8340.4 8340.5
form B(l) form B(2)
1s
Bagyinka et al.
-
*
3d peak area 0.005 eV (re1 to NiC142-)38
SIN
% Ni EPR detectable
% of EPR active Ni poised
0.006 (0.05) 0.015 (0.132) 0.010 (0.088)
106.34 549.48 12.16
85 91 91
80 78 95
8339.4 8339.8
0.040 (0.351) 0.015 (0.132)
409.93 308.38
90 96
85 80
SI
8339.8
0.014 (0.123)
465.24
0
(100)
form C ( l ) form C(2)
8339.3 8339.6
20.56 666.15
80 80
100 100
R
8339.5
1018.21
0
(100)
