The H2 Sensor of Ralstonia eutropha
2001; Elsevier BV; Volume: 276; Issue: 19 Linguagem: Inglês
10.1074/jbc.m009802200
ISSN1083-351X
AutoresMichael Bernhard, Thorsten Buhrke, Boris Bleijlevens, António L. De Lacey, Vı́ctor M. Fernández, Simon P. J. Albracht, Bärbel Friedrich,
Tópico(s)Microbial Fuel Cells and Bioremediation
ResumoPrevious genetic studies have revealed a multicomponent signal transduction chain, consisting of an H2 sensor, a histidine protein kinase, and a response regulator, which controls hydrogenase gene transcription in the proteobacterium Ralstonia eutropha. In this study, we isolated the H2 sensor and demonstrated that the purified protein forms a complex with the histidine protein kinase. Biochemical and spectroscopic analysis revealed that the H2 sensor is a cytoplasmic [NiFe]-hydrogenase with unique features. The H2-oxidizing activity was 2 orders of magnitude lower than that of standard hydrogenases and insensitive to oxygen, carbon monoxide, and acetylene. Interestingly, only H2 production but no HD formation was detected in the D2/H+ exchange assay. Fourier transform infrared data showed an active site similar to that of standard [NiFe]-hydrogenases. It is suggested that the protein environment accounts for a restricted gas diffusion and for the typical kinetic parameters of the H2 sensor. EPR analysis demonstrated that the [4Fe-4S] clusters within the small subunit were not reduced under hydrogen even in the presence of dithionite. Optical spectra revealed the presence of a novel, redox-active, n = 2 chromophore that is reduced by H2. The possible involvement of this chromophore in signal transduction is discussed. Previous genetic studies have revealed a multicomponent signal transduction chain, consisting of an H2 sensor, a histidine protein kinase, and a response regulator, which controls hydrogenase gene transcription in the proteobacterium Ralstonia eutropha. In this study, we isolated the H2 sensor and demonstrated that the purified protein forms a complex with the histidine protein kinase. Biochemical and spectroscopic analysis revealed that the H2 sensor is a cytoplasmic [NiFe]-hydrogenase with unique features. The H2-oxidizing activity was 2 orders of magnitude lower than that of standard hydrogenases and insensitive to oxygen, carbon monoxide, and acetylene. Interestingly, only H2 production but no HD formation was detected in the D2/H+ exchange assay. Fourier transform infrared data showed an active site similar to that of standard [NiFe]-hydrogenases. It is suggested that the protein environment accounts for a restricted gas diffusion and for the typical kinetic parameters of the H2 sensor. EPR analysis demonstrated that the [4Fe-4S] clusters within the small subunit were not reduced under hydrogen even in the presence of dithionite. Optical spectra revealed the presence of a novel, redox-active, n = 2 chromophore that is reduced by H2. The possible involvement of this chromophore in signal transduction is discussed. membrane-bound hydrogenase dichlorophenolindophenol Fourier transform infrared regulatory hydrogenase soluble hydrogenase polyacrylamide gel electrophoresis 4-morpholineethanesulfonic acid 4-morpholinepropanesulfonic acid The detection of physiologically important gases by organisms is mediated by biological sensors that convert the molecular signal into a cellular response. Sensors for O2, CO, and NO have been described, and the signaling mechanism is the subject of current research (1Ignarro L.J. Degnan J.N. Baricos W.H. Kadowitz P.J. Wolin M.S. Biochim. Biophys. Acta. 1982; 718: 49-59Crossref PubMed Scopus (223) Google Scholar, 2Shelver D. Kerby R.L. He Y. Roberts G.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11216-11220Crossref PubMed Scopus (192) Google Scholar, 3Gilles-Gonzalez M.A. Ditta G.S. Helinski D.R. Nature. 1991; 350: 170-172Crossref PubMed Scopus (417) Google Scholar). One of the best studied examples is the two-component FixL-FixJ system of Rhizobium meliloti andBradyrhizobium japonicum. In this case the presence of O2 is detected by a heme-containing histidine protein kinase (4Gilles-Gonzalez M.A. Gonzalez G. Perutz M.F. Biochemistry. 1995; 34: 232-236Crossref PubMed Scopus (125) Google Scholar). The heme group in FixL binds the oxygen molecule that induces a transition of the ferrous iron from high spin to low spin. This triggers the inactivation of the kinase domain of FixL. The release of O2, at low O2 tensions, restores the S = 2 state of the heme iron, which in turn leads to activation of the kinase by autophosphorylation. Subsequent phosphoryl transfer to the response activator FixJ finally stimulates gene transcription (5Gong W. Hao B. Mansy S.S. Gonzalez G. Gilles-Gonzalez M.A. Chan M.K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15177-15182Crossref PubMed Scopus (344) Google Scholar). In the facultative chemolithotrophic proteobacterium Ralstonia eutropha (formerly Alcaligenes eutrophus), the oxidation of molecular hydrogen is catalyzed by two [NiFe]-hydrogenases, a membrane-bound (MBH)1 and a cytoplasmic NAD-reducing hydrogenase (SH) (6Schink B. Schlegel H.G. Biochim. Biophys. Acta. 1979; 567: 315-324Crossref PubMed Scopus (157) Google Scholar, 7Schneider K. Schlegel H.G. Biochim. Biophys. Acta. 1976; 452: 66-80Crossref PubMed Scopus (277) Google Scholar). The structural genes of both [NiFe]-hydrogenases together with sets of accessory genes are grouped in the MBH and SH operons, which are induced in the presence of molecular hydrogen (8Schwartz E. Gerischer U. Friedrich B. J. Bacteriol. 1998; 180: 3197-3204Crossref PubMed Google Scholar, 9Lenz O. Friedrich B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12474-12479Crossref PubMed Scopus (151) Google Scholar). Hydrogenase gene transcription is controlled by a multicomponent regulatory system consisting of the proteins HoxB, HoxC, HoxJ, and HoxA, which are encoded in the MBH operon (8Schwartz E. Gerischer U. Friedrich B. J. Bacteriol. 1998; 180: 3197-3204Crossref PubMed Google Scholar, 9Lenz O. Friedrich B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12474-12479Crossref PubMed Scopus (151) Google Scholar, 10Lenz O. Strack A. Tran-Betcke A. Friedrich B. J. Bacteriol. 1997; 179: 1655-1663Crossref PubMed Google Scholar). HoxJ and HoxA share typical features of a bacterial two-component regulatory system that recognizes and responds to various environmental stimuli (9Lenz O. Friedrich B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12474-12479Crossref PubMed Scopus (151) Google Scholar, 11Stock J.B. Surette M.G. Levit M. Park P. Hoch J.A. Silhavy T.J. Two-component Signal Transduction. American Society for Microbiology, Washington, D. C.1995: 25-51Google Scholar). Our studies showed that HoxJ displays autokinase activity (9Lenz O. Friedrich B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12474-12479Crossref PubMed Scopus (151) Google Scholar) and communicates with the activator HoxA, 2M. Forgber, O. Lenz, E. Schwartz, and B. Friedrich, unpublished results. a member of the NtrC family of response regulators (12Eberz G. Friedrich B. J. Bacteriol. 1991; 173: 1845-1854Crossref PubMed Google Scholar). HoxA, the final target of the H2-sensing signal transduction chain, binds to the upstream region of the hydrogenase promoters and activates open complex formation by ς54 RNA polymerase (8Schwartz E. Gerischer U. Friedrich B. J. Bacteriol. 1998; 180: 3197-3204Crossref PubMed Google Scholar, 13Zimmer D. Schwartz E. Tran-Betcke A. Gewinner P. Friedrich B. J. Bacteriol. 1995; 177: 2373-2380Crossref PubMed Google Scholar). Genetic studies revealed that recognition of H2 requires in addition to HoxA and HoxJ the protein HoxBC (9Lenz O. Friedrich B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12474-12479Crossref PubMed Scopus (151) Google Scholar). Proteins similar to HoxBC, designated HupUV, have been identified in Rhodobacter capsulatus and B. japonicum (14Elsen S. Colbeau A. Chabert J. Vignais P.M. J. Bacteriol. 1996; 178: 5174-5181Crossref PubMed Google Scholar, 15Black L.K. Fu C. Maier R.J. J. Bacteriol. 1994; 176: 7102-7106Crossref PubMed Google Scholar). HoxBC-like proteins show typical features of a [NiFe]-hydrogenase (16Kleihues L. Lenz O. Bernhard M. Buhrke T. Friedrich B. J. Bacteriol. 2000; 182: 2716-2724Crossref PubMed Scopus (113) Google Scholar). Although HoxBC is essential for lithoautotrophic growth of R. eutropha (9Lenz O. Friedrich B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12474-12479Crossref PubMed Scopus (151) Google Scholar), it cannot compensate for the loss of the MBH and the SH. This observation points to a regulatory rather than an energy-yielding function of the HoxBC protein (16Kleihues L. Lenz O. Bernhard M. Buhrke T. Friedrich B. J. Bacteriol. 2000; 182: 2716-2724Crossref PubMed Scopus (113) Google Scholar). The low level of expression combined with an extremely low activity allowed only preliminary biochemical analysis of HoxBC in crude extracts (17Pierik A. Schmelz M. Lenz O. Friedrich B. Albracht S.P. FEBS Lett. 1998; 438: 231-235Crossref PubMed Scopus (57) Google Scholar). Attempts to express a functional HoxBC protein in Escherichia coli were unsuccessful. This prompted us to develop a homologous overexpression system in R. eutropha (16Kleihues L. Lenz O. Bernhard M. Buhrke T. Friedrich B. J. Bacteriol. 2000; 182: 2716-2724Crossref PubMed Scopus (113) Google Scholar) and to use it successfully for the purification of HoxBC, later named regulatory hydrogenase (RH). Biochemical and spectroscopic analysis of the homogenous RH uncovered unique enzymatic features that are clearly distinct from the properties of standard hydrogenases. The data suggest that the RH shows a common [NiFe] active site but displays significant changes in the protein environment. In order to study the mechanism of H2 signal transduction in more depth, we began to establish an in vitro system, using purified components. First data show that the RH forms a specific complex with the sensor kinase HoxJ, supporting the notion that the RH is a direct component of the signal transduction chain. R. eutropha strain HF371, a derivative of R. eutropha H16, harboring plasmid pGE378, was used for protein purification (16Kleihues L. Lenz O. Bernhard M. Buhrke T. Friedrich B. J. Bacteriol. 2000; 182: 2716-2724Crossref PubMed Scopus (113) Google Scholar). Cells were heterotrophically grown in a mineral medium in a 10-liter Braun Biostat fermentor (Braun, Melsungen, Germany) at 30 °C under hydrogenase derepressing conditions. At an OD436 of 11 the cells were harvested, washed in 50 mm potassium phosphate buffer, pH 7.0 (K-PO4 buffer), and stored frozen in liquid nitrogen. Cells (83 g, wet weight) were resuspended in 30 ml of K-PO4 buffer containing 0.1 mmphenylmethylsulfonyl fluoride. Cells were disrupted by two passages through a chilled Amicon French press cell at 1100 pounds/square inch (75.8 bar). Soluble extracts were prepared by high speed centrifugation (100 000 × g, 60 min, 4 °C). The resulting supernatant was degassed and saturated with hydrogen. The extract was kept under an atmosphere of 100% H2 and subsequently incubated for 10 min at 65 °C. After the heat treatment the sample was chilled on ice. All further purification steps were carried out under air. The denatured proteins were removed by centrifugation (13 000 × g, 20 min, 4 °C), and the supernatant was fractionated by addition of (NH4)2SO4 to give a final concentration of 1 m. The precipitated proteins were removed by centrifugation (13 000 × g, 20 min, 4 °C), and the clear supernatant was directly applied to a POROS 20ET column (Applied Biosystems; ethyl ether; 10 × 100 mm), preequilibrated with K-PO4 buffer containing 1m (NH4)2SO4 at a flow rate of 40 ml/min (BioCAD Perfusion Chromatography Workstation). The column was washed with 2 bed volumes of K-PO4 buffer containing 1 m(NH4)2SO4. The protein was eluted with K-PO4 buffer containing 0.4 m(NH4)2SO4, and fractions of 4 ml were collected. The active fractions of several column runs were combined, concentrated, and dialyzed against K-PO4 buffer. The RH was further purified on a POROS 20HQ column (Applied Biosystems; quarternized polyethyleneimine; 4.6 × 100 mm) preequilibrated with K-PO4 buffer. The eluent was pooled, concentrated (Centriprep-10; Amicon), and directly frozen in liquid N2. Protein concentrations were determined according to the methods of Lowry et al. (18Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Proteins were resolved by electrophoresis in 12% polyacrylamide/SDS gels and transferred to Protran BA85 nitrocellulose membranes (Schleicher & Schüll). HoxC was detected with anti-HoxC serum, diluted 1:1000, and an alkaline-phosphatase-labeled goat anti-rabbit IgG (Dianova, Hamburg, Germany). The histidine protein kinase HoxJ was overproduced in E. coli and purified as a polyhistidine-tagged protein, His6-HoxJ, by metal chelate affinity chromatography (9Lenz O. Friedrich B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12474-12479Crossref PubMed Scopus (151) Google Scholar). Purified His6-HoxJ and RH were mixed and subsequently applied to a native 4–15% polyacrylamide gel. Native gel electrophoresis was carried out as described previously (19Bernhard M. Schwartz E. Rietdorf J. Friedrich B. J. Bacteriol. 1996; 178: 4522-4529Crossref PubMed Google Scholar). Complex formation was either monitored by in-gel hydrogenase activity staining (19Bernhard M. Schwartz E. Rietdorf J. Friedrich B. J. Bacteriol. 1996; 178: 4522-4529Crossref PubMed Google Scholar) or by protein staining using Coomassie Blue. Nickel and iron were determined with a Hitachi 180-80 polarized Zeeman atomic absorption spectrophotometer against a standard series. Samples were made devoid of extraneous metal ions by passage over a Chelex-100 column (Bio-Rad). Hydrogen uptake activity was measured amperometrically at 30 °C in a cell (2.15 ml) with 50 mmTris-HCl, pH 8.0, using a Clark-type electrode (YSI 5331) according to Coremans et al. (20Coremans J.M.C.C. Van der Zwaan J.W. Albracht S.P.J. Biochim. Biophys. Acta. 1989; 997: 256-267Crossref Scopus (48) Google Scholar). As O2 did not affect the activity, no efforts were made to remove air. Hydrogen, in the form of H2-saturated water, was added to final concentrations varying from 36 to 100 μm. As electron acceptor either benzyl viologen (4.2 mm, Em = -359 mV) or methylene blue (4 mm,Em, pH 7 = +11 mV) were used. The measured specific activities were plotted against the H2concentration. The dependence was simulated using the program Leonora by Cornish-Bowden, assuming Michaelis-Menten kinetics (21Cornish-Bowden A. Analysis of Enzyme Kinetic Data. Oxford University Press, New York1995: 133-189Google Scholar). Protein concentrations in the assay were typically 2.5–5 nm RH (α2β2). Benzyl viologen-dependent H2 evolution was determined amperometrically at 30 °C. The reaction mixture contained 50 mm acetate buffer, 1 mm benzyl viologen, and 3 mm sodium dithionite. D+/H+ exchange activity was measured in a stirred membrane leak chamber fitted to a mass spectrometer (Masstorr 200 DX quadrupole, VG Quadrupoles Ltd.). Two different assays were used. In the first assay 10 ml of Mes/Mops/Tris buffer solution (ionic strength 90 mm; pH 6.5) was saturated with 20% D2 and 80% argon, and 1 μmol of sodium dithionite was added to eliminate residual oxygen. The reaction was started by the addition of RH (α2β2) to a final concentration of 0.12 μm. Masses 1–6 were scanned at 1 atomic mass unit/s. In the second assay the buffer solution was in 99.9% D2O (Aldrich) and saturated with H2. A control experiment was done in D2/D2O in order to evaluate the HD production catalyzed by the protein due to contaminant H+. This effect was subtracted to the H2/D2O assay. The pD of the assay mixtures was measured with a glass electrode calibrated with pH standards in H2O. It was taken into account that pD = pH + 0.41 (22Covington A.K. Paabo M. Robinson R.A. Bates R.G. Anal. Chem. 1968; 40: 700-706Crossref Scopus (619) Google Scholar, 23Quinn D.M. Sutton L.D. Cook P.F. Enzyme Mechanism from Isotope Effects. CRC, Boca Raton, FL1991: 73-126Google Scholar). All experiments were done at 30 °C. X-band (9.4 GHz) spectra with a 100 kHz field modulation frequency were recorded on a Bruker ECS106 EPR spectrometer equipped with an Oxford Instruments ESR900 helium flow cryostat with an ITC4 temperature controller. The magnetic field was calibrated with an AEG Magnetic Field Meter. The frequency was measured with a Hewlett-Packard 5350B Microwave Frequency Counter. Illumination of the samples was performed by shining white light (Osram Halogen Bellaphot, 150 watts) via a light guide through the irradiation grid of the Bruker ER 4102 ST cavity. Spectra were simulated according to the formulas published by Beinert and Albracht (24Beinert H. Albracht S.P. Biochim. Biophys. Acta. 1982; 683: 245-277Crossref PubMed Scopus (148) Google Scholar). Fourier transform infrared (FTIR) spectra were taken on a Bio-Rad FTS 60A spectrometer equipped with an MCT detector. Spectra were recorded at room temperature with a resolution of 2 cm−1. Typically, averages of 1524 spectra were taken against proper blanks. Enzyme samples (10 μl) were loaded into a gas-tight transmission cell (CaF2, 56 μm path length). The spectra were corrected for the base line using a spline function provided by the Bio-Rad software. Optical spectra were taken on an Aminco DW-2000 spectrophotometer interfaced with an IBM computer. To avoid interferences with the dominant activities of the MBH and the SH, we started the purification of the RH protein with mutant R. eutrophaHF371, in which the MBH and SH genes had been deleted by mutation. After cell disruption and high speed centrifugation, the soluble extract was incubated at 65 °C for 10 min under an atmosphere of H2 (100%). The heat treatment was necessary prior to high resolution hydrophobic interaction chromatography to provide an effective and rapid isolation of the RH. Purification to apparent homogeneity was achieved by subsequent anion exchange chromatography. The total procedure is summarized in TableI. Beginning with 83 g of cells (wet weight), 10.9 mg of RH was obtained. The specific activity of the preparation was 0.94 units/mg of protein with methylene blue as the electron acceptor. The H2 concentration in the assay was 57.8 μm. The protein was purified 26-fold with a yield of 6%.Table IPurification of the R. eutropha regulatory hydrogenaseStepaThe individual steps are described in detail under "Experimental Procedures."Total proteinTotal activitySpecific activityRecoveryPurificationmgunitsunits/mg%-foldSoluble extract4676168.50.036100Heat treatment173483.30.04849.41.3(NH4)2SO4162079.40.04947.11.4ET column34.823.40.67313.918.7HQ column10.910.30.9426.126.2a The individual steps are described in detail under "Experimental Procedures." Open table in a new tab Two protein bands occurred after denaturing the RH by SDS-PAGE corresponding to molecular masses of 37 and 55 kDa, respectively (Fig.1 A). These values are in good agreement with those predicted from the nucleotide sequence ofhoxB (36.5 kDa) and hoxC (52.4 kDa). The identity of the purified protein was confirmed by immunoblot analysis, using an antibody raised against the HoxC subunit of the RH (Fig.1 B). Analysis of the enzyme on a Superdex G-200 (Amersham Pharmacia Biotech) revealed a single peak corresponding to a molecular mass of ∼165 kDa (data not shown) indicating that the RH was purified as a tetramer with an α2β2 structure. Atomic absorption spectroscopy showed an average metal content of 11.2 iron/nickel. After Chelex treatment this ratio decreased to 7.6 iron/nickel. The activity after the Chelex-100 column was 75% of the initial activity. The oxidation of H2 by the purified RH turned out to be O2-insensitive. The level of activity was the same in aerobic and anaerobic buffers. Moreover, the rate of H2oxidation determined with methylene blue as the electron acceptor did not show the typical lag phase that is found with most as isolated [NiFe]-hydrogenases. This observation is consistent with the result obtained with soluble extract (17Pierik A. Schmelz M. Lenz O. Friedrich B. Albracht S.P. FEBS Lett. 1998; 438: 231-235Crossref PubMed Scopus (57) Google Scholar). The Km for H2 was 25 ± 5 μm, and the calculated specific activity at Vmax conditions was 1.2 ± 0.2 units/mg of protein. The activity of the RH remained constant over a broad pH range between 5 and 10 irrespective of the used buffers (potassium acetate, K-PO4, and Tris-HCl; 50 mm each), whereas most hydrogenases show a distinct pH optimum. In contrast to the H2 uptake activity, the production of H2 by the RH was pH-dependent. Highest H2 evolution rates (0.8 units/mg of protein) were obtained at pH 4.0 with benzyl viologen as electron donor. Acetylene has been shown to be a competitive inhibitor for several hydrogenases (25Hyman M.R. Arp D.J. Biochemistry. 1987; 26: 6447-6454Crossref Scopus (18) Google Scholar, 26Zorin N.A. Dimon B. Gagnon J. Gaillard J. Carrier P. Vignais P.M. Eur. J. Biochem. 1996; 241: 675-681Crossref PubMed Scopus (51) Google Scholar). Incubation of the RH with C2H2 did not affect the RH activity (data not shown). Storage of the purified RH at 4 °C under air or an atmosphere of 100% O2 resulted in a loss of 50% of the H2-dependent methylene blue-reducing activity within 48 h. Replacement of the air atmosphere by 100% argon or N2 caused a decrease of 20% of the activity within the same period. Addition of metal ions (Fe3+, Ni2+, Mn2+, Mg2+, and Zn2+) or glycerol (20%) or addition of KCl up to 0.5m did not affect the stability of the RH. The supply of dithionite or ferricyanide under anoxic conditions also did not contribute to the stability of the RH. Moreover, storage of the isolated RH under an atmosphere of 100% H2 inactivated the RH rapidly; 50% of its activity disappeared within 12 h. The H2 sensitivity contrasts with data obtained with the soluble extract, which showed constant RH activity over a period of 24 h under comparable conditions. In all cases inactivation of the RH was irreversible. The D+/H+ exchange assay with the RH yielded only H2 production but no HD formation (Fig.2 A). The initial rate of H2 production at pH 6.5 was 2.1 ± 0.1 units/mg of protein. This behavior is distinct from that of other [NiFe]-hydrogenases, which show higher initial rates of HD production than of H2 production. When the exchange activity assay was measured in deuterated water saturated with H2 some HD production was detected with the RH, although the rate of D2 evolution was definitively higher (Fig. 2 B). The initial rate of D2 production at pD 6.5 was 1.3 ± 0.2 units/mg of protein, whereas the initial rate of HD production was 0.5 ± 0.1 units/mg of protein. The pH optimum of the D+/H+ exchange activity of the RH was at pH 5.5 (data not shown). Preliminary studies of the RH in crude cell extracts prohibited a study of the EPR properties of its Fe-S clusters (17Pierik A. Schmelz M. Lenz O. Friedrich B. Albracht S.P. FEBS Lett. 1998; 438: 231-235Crossref PubMed Scopus (57) Google Scholar). The purified enzyme now allowed this approach. The as isolated RH showed no EPR signals at temperatures between 4.2 and 100 K. Also after addition of the oxidizing agent DCIP (dichlorophenolindophenol, Em = +230 mV), no signal occurred. Upon reduction of the RH (15 min under 100% H2at room temperature in 50 mm Tris-HCl, pH 8.0), a rhombic EPR signal with g values at 2.19, 2.13, and 2.01 appeared (Fig.3, trace A). The double-integrated intensity of the signal amounted to a spin concentration equal to 69% of the nickel concentration. The EPR signal is very similar to the well studied Nia-C* signal observed in standard hydrogenases (e.g. fromDesulfovibrio gigas and Allochromatium vinosum), and it is due to a paramagnetic state of the active site nickel in the 3+ state (27Happe R.P. Roseboom W. Albracht S.P. Eur. J. Biochem. 1999; 259: 602-608Crossref PubMed Scopus (65) Google Scholar). A typical feature of enzyme in the Nia-C* state is its light sensitivity at cryogenic temperatures, yielding the so-called Nia-L* signal as a result of the photodissociation of a hydrogen (28Van der Zwaan J.W. Albracht S.P.J. Fontijn R.D. Mul P. Eur. J. Biochem. 1987; 169: 377-384Crossref PubMed Scopus (39) Google Scholar). A model for this photodissociation has been described by Happe et al.(27Happe R.P. Roseboom W. Albracht S.P. Eur. J. Biochem. 1999; 259: 602-608Crossref PubMed Scopus (65) Google Scholar). In the case of the RH the Nia-C* signal also showed this light-sensitive behavior. Upon illumination at 30 K a spectrum (Nia-L*: gxyz = 2.045, 2.09, and 2.24), only slightly different from the Nia-L* signal of standard hydrogenases, appeared (Fig. 3, trace B). The small difference concerns the position of the gz (2.24 in the RH as compared with 2.28/2.30 in standard [NiFe] hydrogenases). This points to a small structural difference around the active site nickel. Upon warming of the sample to 200 K for 15 min in the dark a third, transient, spectrum came up with g values at 2.047, 2.069, and 2.30 (Fig. 3, trace C). Only after several hours at 200 K the sample returned to the Nia-C* state. Contrary to observations in standard [NiFe]-hydrogenases no signal of a [3Fe-4S]+ cluster could be observed in the oxidized protein, not even after treatment with excess DCIP. This is in agreement with the presence of three [4Fe-4S] clusters as predicted from the amino acid sequence data. When the protein was treated with 100% H2, however, no signals due to reduced cubanes were detectable, not even if 20 mm dithionite was added. None of the nickel signals (Nia-C*, Nia-L*, or the transient signal) showed any spin coupling due to a reduced proximal [4Fe-4S] cluster (Fig. 3). This indicates that this cluster was in the oxidized, diamagnetic state in the RH under H2. In standard [NiFe]-hydrogenases the proximal cluster is usually reduced under 100% H2. The interaction of the nickel with the reduced proximal cluster is observed as a clear 2-fold splitting of the Nia-C* signal at 4.5 K. At low temperatures it was also possible to completely saturate the Nia-C* signal at high microwave power (260 milliwatts), which is again indicative of an oxidized proximal cluster (28Van der Zwaan J.W. Albracht S.P.J. Fontijn R.D. Mul P. Eur. J. Biochem. 1987; 169: 377-384Crossref PubMed Scopus (39) Google Scholar, 29Teixeira M. Fauque G. Moura I. Lespinat P.A. Berlier Y. Prickril B. Peck Jr., H.D. Xavier A.V. LeGall J. Moura J.J.G. Eur. J. Biochem. 1987; 167: 47-58Crossref PubMed Scopus (130) Google Scholar). Reduction of the RH with dithionite in the presence or absence of low potential electron acceptors (methyl viologen and benzyl viologen) under 100% H2 did not evoke any signal of a reduced Fe-S cluster. Also inspection of the integrated EPR signals did not uncover any broad signal due to reduced Fe-S clusters as can be seen in the right-hand panel in Fig. 3for the Nia-L* signal. The FTIR measurements on purified RH confirmed the presence of only two redox states described earlier to be present in the RH from soluble extracts (17Pierik A. Schmelz M. Lenz O. Friedrich B. Albracht S.P. FEBS Lett. 1998; 438: 231-235Crossref PubMed Scopus (57) Google Scholar). Untreated protein showed a spectrum (Fig. 4 A) with two small bands (2082 and 2071 cm−1) and one large band (1943 cm−1) in the 2150–1850 cm−1 spectral region. This EPR-silent state of the active RH resembles the Nia-S state of standard [NiFe]-hydrogenases. Maximal reduction, already obtained after a few minutes under 100% H2 at room temperature, yielded the Nia-C* state (Fig. 4 C) as identified previously in other [NiFe]-hydrogenases (30Bagley K.A. Duin E.C. Roseboom W. Albracht S.P. Woodruff W.H. Biochemistry. 1995; 34: 5527-5535Crossref PubMed Scopus (199) Google Scholar, 31De Lacey A.L. Hatchikian E.C. Volbeda A. Frey M. Fontecilla-Camps J.C. Fernandez V.M. J. Am. Chem. Soc. 1997; 119: 7181-7189Crossref Scopus (258) Google Scholar). This state showed a CO stretch vibration at 1960 cm−1. The two bands at 2082 and 2071 cm−1, which did not shift, are ascribed to the symmetrical and antisymmetrical coupled vibrations of two cyanides bound to iron in the active site (17Pierik A. Schmelz M. Lenz O. Friedrich B. Albracht S.P. FEBS Lett. 1998; 438: 231-235Crossref PubMed Scopus (57) Google Scholar). It was not possible to reduce further this state by adding excess dithionite (20 mm, spectrum not shown). When the gas phase was changed from 100% H2 to 100% CO (equilibration time 60 min) a mixture of the Nia-C* and Nia-S state was observed (Fig. 4 B). The spectrum clearly showed that it was not possible for exogenous CO to bind to the active site of the RH since no extra peak around 2060 cm−1 could be seen. Such a band from added CO is observed in the A. vinosum and D. gigasenzyme 3De Lacey, A.L., and Fernandez, V.M., unpublished results. (32Bagley K.A. Van Garderen C.J. Chen M. Duin E.C. Albracht S.P. Woodruff W.H. Biochemistry. 1994; 33: 9229-9236Crossref PubMed Scopus (135) Google Scholar). A similar change was observed by replacing H2 with argon (results not shown). Upon complete oxidation with excess DCIP (2 mm) the sample returned to the Nia-S state. UV-visible spectra of oxidized and reduced RH showed differences in absorption between the two species. Incubation of the RH under 100% H2 resulted in an increase in absorption in the 250–280 and 300–400 nm spectral regions (Fig. 5). The difference spectrum of reduced minus oxidized RH showed a large peak at 251 nm and a smaller one at 342 nm with an apparent shoulder at 305 nm. The calculated ε251 was 11.96 mm−1 cm−1based on protein concentration. Similarly the ε342 was calculated to be 5.36 mm−1cm−1. The protein concentration used (0.64 mg/ml) was such that the absorption at 280 nm was about 1.0. At this intensity the detector is still sensitive enough to pick up reliable differences in the UV, meaning that these are not due to mismatching in this region. To elucidate the nature of the interaction between the RH and the signal
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