Reduction of Unusual Iron-Sulfur Clusters in the H2-sensing Regulatory Ni-Fe Hydrogenase from Ralstonia eutropha H16
2005; Elsevier BV; Volume: 280; Issue: 20 Linguagem: Inglês
10.1074/jbc.m500601200
ISSN1083-351X
AutoresThorsten Buhrke, Simone Löscher, Oliver Lenz, E. Schlodder, Ingo Zebger, Lars Klembt Andersen, Peter Hildebrandt, Wolfram Meyer‐Klaucke, Holger Dau, Bärbel Friedrich, Michael Haumann,
Tópico(s)Hydrogen Storage and Materials
ResumoThe regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha functions as a hydrogen sensor. The RH consists of the large subunit HoxC housing the Ni-Fe active site and the small subunit HoxB containing Fe-S clusters. The heterolytic cleavage of H2 at the Ni-Fe active site leads to the EPR-detectable Ni-C state of the protein. For the first time, the simultaneous but EPR-invisible reduction of Fe-S clusters during Ni-C state formation was demonstrated by changes in the UV-visible absorption spectrum as well as by shifts of the iron K-edge from x-ray absorption spectroscopy in the wild-type double dimeric RHWT [HoxBC]2 and in a monodimeric derivative designated RHstop lacking the C-terminal 55 amino acids of HoxB. According to the analysis of iron EXAFS spectra, the Fe-S clusters of HoxB pronouncedly differ from the three Fe-S clusters in the small subunits of crystallized standard Ni-Fe hydrogenases. Each HoxBC unit of RHWT seems to harbor two [2Fe-2S] clusters in addition to a 4Fe species, which may be a [4Fe-3S-3O] cluster. The additional 4Fe-cluster was absent in RHstop. Reduction of Fe-S clusters in the hydrogen sensor RH may be a first step in the signal transduction chain, which involves complex formation between [HoxBC]2 and tetrameric HoxJ protein, leading to the expression of the energy converting Ni-Fe hydrogenases in R. eutropha. The regulatory Ni-Fe hydrogenase (RH) from Ralstonia eutropha functions as a hydrogen sensor. The RH consists of the large subunit HoxC housing the Ni-Fe active site and the small subunit HoxB containing Fe-S clusters. The heterolytic cleavage of H2 at the Ni-Fe active site leads to the EPR-detectable Ni-C state of the protein. For the first time, the simultaneous but EPR-invisible reduction of Fe-S clusters during Ni-C state formation was demonstrated by changes in the UV-visible absorption spectrum as well as by shifts of the iron K-edge from x-ray absorption spectroscopy in the wild-type double dimeric RHWT [HoxBC]2 and in a monodimeric derivative designated RHstop lacking the C-terminal 55 amino acids of HoxB. According to the analysis of iron EXAFS spectra, the Fe-S clusters of HoxB pronouncedly differ from the three Fe-S clusters in the small subunits of crystallized standard Ni-Fe hydrogenases. Each HoxBC unit of RHWT seems to harbor two [2Fe-2S] clusters in addition to a 4Fe species, which may be a [4Fe-3S-3O] cluster. The additional 4Fe-cluster was absent in RHstop. Reduction of Fe-S clusters in the hydrogen sensor RH may be a first step in the signal transduction chain, which involves complex formation between [HoxBC]2 and tetrameric HoxJ protein, leading to the expression of the energy converting Ni-Fe hydrogenases in R. eutropha. Ni-Fe hydrogenases represent an important class of metalloenzymes that catalyze the reversible cleavage of molecular hydrogen into electrons and protons (reaction H2 ⇔ 2H+ + 2e-) (1Cammack R. Robson R. Frey M. Hydrogen as a Fuel: Learning from Nature. Taylor & Francis Ltd., London1997Google Scholar). The chemolithoautotrophic β-proteobacterium bacterium Ralstonia eutropha H16 houses three different Ni-Fe hydrogenases that are physiologically active in the presence of O2 (2Schink B. Schlegel H.G. Biochimie (Paris). 1978; 60: 297-305Crossref PubMed Scopus (50) Google Scholar, 3Pierik A.J. Schmelz M. Lenz O. Friedrich B. Albracht S.P. FEBS Lett. 1998; 438: 231-235Crossref PubMed Scopus (58) Google Scholar). The membrane-bound and soluble NAD-reducing enzymes are involved in energy conversion (4Schneider K. Schlegel H.G. Biochim. Biophys. Acta. 1976; 452: 66-80Crossref PubMed Scopus (279) Google Scholar, 5Schink B. Schlegel H.G. Biochim. Biophys. Acta. 1979; 567: 315-324Crossref PubMed Scopus (157) Google Scholar). The regulatory Ni-Fe hydrogenase (RH) 1The abbreviations used are: RH, regulatory Ni-Fe hydrogenase; AAS, atomic absorption spectroscopy; EPR, electron paramagnetic resonance spectroscopy; EXAFS, extended X-ray absorption fine structure; FTIR, Fourier transform (FT) infrared spectroscopy; RH+H2,H2-flushed RH; RHox, air-oxidized RH; TRXFA, total reflection X-ray fluorescence analysis; XANES, X-ray absorption near-edge structure; XAS, X-ray absorption spectroscopy. belongs to a particularly interesting type of Ni-Fe hydrogenase functioning as hydrogen sensors (6Kleihues L. Lenz O. Bernhard M. Buhrke T. Friedrich B. J. Bacteriol. 2000; 182: 2716-2724Crossref PubMed Scopus (116) Google Scholar). Hydrogen sensors have also been described in Bradyrhizobium japonicum (7Black L.K. Fu C. Maier R.J. J. Bacteriol. 1994; 176: 7102-7106Crossref PubMed Google Scholar) and Rhodobacter capsulatus (8Elsen S. Colbeau A. Chabert J. Vignais P.M. J. Bacteriol. 1996; 178: 5174-5181Crossref PubMed Google Scholar). Upon the interaction of the RH with molecular hydrogen, a complex signal transduction cascade is initiated that leads to the expression of the energy-converting hydrogenases (9Lenz O. Friedrich B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12474-12479Crossref PubMed Scopus (153) Google Scholar). The RH consists of the large subunit HoxC that harbors the hydrogen-activating Ni-Fe site and the small subunit HoxB that contains iron-sulfur clusters (6Kleihues L. Lenz O. Bernhard M. Buhrke T. Friedrich B. J. Bacteriol. 2000; 182: 2716-2724Crossref PubMed Scopus (116) Google Scholar). Several unusual properties of the RH (3Pierik A.J. Schmelz M. Lenz O. Friedrich B. Albracht S.P. FEBS Lett. 1998; 438: 231-235Crossref PubMed Scopus (58) Google Scholar, 10Bernhard M. Buhrke T. Bleijlevens B. De Lacey A.L. Fernandez V.M. Albracht S.P. Friedrich B. J. Biol. Chem. 2001; 276: 15592-15597Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 11Haumann M. Porthun A. Buhrke T. Liebisch P. Meyer-Klaucke W. Friedrich B. Dau H. Biochemistry. 2003; 42: 11004-11015Crossref PubMed Scopus (37) Google Scholar) are remarkably different from those of the so-called standard Ni-Fe hydrogenases from inter alia, Desulfovibrio gigas and Allochromatium vinosum (1Cammack R. Robson R. Frey M. Hydrogen as a Fuel: Learning from Nature. Taylor & Francis Ltd., London1997Google Scholar, 12Frey M. J. Chem. Biochem. 2002; 3: 153-160Google Scholar, 13Albracht S.P.J. Biochim. Biophys. Acta. 1994; 1188: 167-204Crossref PubMed Scopus (453) Google Scholar). In contrast to the dimeric standard Ni-Fe hydrogenases, the RH forms a double dimer [HoxBC]2 (Fig. 1A) that is connected to a tetramer of the HoxJ protein (14Buhrke T. Lenz O. Porthun A. Friedrich B. Mol. Microbiol. 2004; 51: 1677-1689Crossref PubMed Scopus (56) Google Scholar). The N-terminal input module of HoxJ containing a PAS domain is required for the formation of the RH-HoxJ complex, whereas the C-terminal domain of HoxJ has histidine protein kinase activity (14Buhrke T. Lenz O. Porthun A. Friedrich B. Mol. Microbiol. 2004; 51: 1677-1689Crossref PubMed Scopus (56) Google Scholar). The RH cleaves H2 only at extremely low rates (3Pierik A.J. Schmelz M. Lenz O. Friedrich B. Albracht S.P. FEBS Lett. 1998; 438: 231-235Crossref PubMed Scopus (58) Google Scholar, 10Bernhard M. Buhrke T. Bleijlevens B. De Lacey A.L. Fernandez V.M. Albracht S.P. Friedrich B. J. Biol. Chem. 2001; 276: 15592-15597Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In contrast to standard hydrogenases, which can exist in up to nine different redox states (1Cammack R. Robson R. Frey M. Hydrogen as a Fuel: Learning from Nature. Taylor & Francis Ltd., London1997Google Scholar, 13Albracht S.P.J. Biochim. Biophys. Acta. 1994; 1188: 167-204Crossref PubMed Scopus (453) Google Scholar), in the RH only two states of functional relevance have been detected (10Bernhard M. Buhrke T. Bleijlevens B. De Lacey A.L. Fernandez V.M. Albracht S.P. Friedrich B. J. Biol. Chem. 2001; 276: 15592-15597Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). After aerobic isolation the enzyme is in its oxidized state containing Ni(II). This state does not need to be activated but is always ready to bind hydrogen, a prerequisite for the sensor function (10Bernhard M. Buhrke T. Bleijlevens B. De Lacey A.L. Fernandez V.M. Albracht S.P. Friedrich B. J. Biol. Chem. 2001; 276: 15592-15597Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 11Haumann M. Porthun A. Buhrke T. Liebisch P. Meyer-Klaucke W. Friedrich B. Dau H. Biochemistry. 2003; 42: 11004-11015Crossref PubMed Scopus (37) Google Scholar). In the presence of H2 it is rapidly converted to a state revealing a typical EPR-signal, termed Ni-C, due to a Ni(III)-H- species formed during heterolytic H2 cleavage (15Brecht M. Van Gastel M. Buhrke T. Friedrich B. Lubitz W. J. Am. Chem. Soc. 2003; 125: 13075-13083Crossref PubMed Scopus (224) Google Scholar). In standard Ni-Fe hydrogenases the nickel is coordinated by four conserved cysteine residues. X-ray absorption spectroscopy (XAS) investigations on the RH, however, revealed that nickel may be coordinated by less than four cysteines (11Haumann M. Porthun A. Buhrke T. Liebisch P. Meyer-Klaucke W. Friedrich B. Dau H. Biochemistry. 2003; 42: 11004-11015Crossref PubMed Scopus (37) Google Scholar). The iron atom of the RH active site, on the other hand, carries two cyanides and one CO molecule, similar to standard Ni-Fe hydrogenases (3Pierik A.J. Schmelz M. Lenz O. Friedrich B. Albracht S.P. FEBS Lett. 1998; 438: 231-235Crossref PubMed Scopus (58) Google Scholar, 10Bernhard M. Buhrke T. Bleijlevens B. De Lacey A.L. Fernandez V.M. Albracht S.P. Friedrich B. J. Biol. Chem. 2001; 276: 15592-15597Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Although information has become available about the sequence of events that occur at the Ni-Fe active site upon interaction of the RH with H2 (10Bernhard M. Buhrke T. Bleijlevens B. De Lacey A.L. Fernandez V.M. Albracht S.P. Friedrich B. J. Biol. Chem. 2001; 276: 15592-15597Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 11Haumann M. Porthun A. Buhrke T. Liebisch P. Meyer-Klaucke W. Friedrich B. Dau H. Biochemistry. 2003; 42: 11004-11015Crossref PubMed Scopus (37) Google Scholar, 15Brecht M. Van Gastel M. Buhrke T. Friedrich B. Lubitz W. J. Am. Chem. Soc. 2003; 125: 13075-13083Crossref PubMed Scopus (224) Google Scholar), it is unclear whether electron transfer out of the Ni-Fe site takes place during H2 cleavage and to where these electrons are transferred. Information on these points is expected to contribute to the understanding of the H2-sensing mechanism of the RH-HoxJ complex (14Buhrke T. Lenz O. Porthun A. Friedrich B. Mol. Microbiol. 2004; 51: 1677-1689Crossref PubMed Scopus (56) Google Scholar). In standard Ni-Fe hydrogenases of the D. gigas type, the small subunit contains three Fe-S clusters, two [4Fe-4S] and one [3Fe-4S] (16Volbeda A. Charon M.H. Piras C. Hatchikian E.C. Frey M. Fontecilla-Camps J.C. Nature. 1995; 373: 556-557Crossref PubMed Scopus (1404) Google Scholar), which are bound via conserved cysteines and one histidine residue found in all Ni-Fe hydrogenase sequences. During hydrogen turnover these clusters become reduced as detected by EPR spectroscopy (1Cammack R. Robson R. Frey M. Hydrogen as a Fuel: Learning from Nature. Taylor & Francis Ltd., London1997Google Scholar, 17Armstrong F.A. Curr. Opin. Chem. Biol. 2004; 8: 133-140Crossref PubMed Scopus (179) Google Scholar). The RH small subunit HoxB also contains these conserved cysteines. Therefore, it was postulated that it might also harbor three Fe-S clusters (6Kleihues L. Lenz O. Bernhard M. Buhrke T. Friedrich B. J. Bacteriol. 2000; 182: 2716-2724Crossref PubMed Scopus (116) Google Scholar). EPR investigation of the RH, however, did not show reduced Fe-S clusters when the Ni-C EPR signal was formed under H2 (3Pierik A.J. Schmelz M. Lenz O. Friedrich B. Albracht S.P. FEBS Lett. 1998; 438: 231-235Crossref PubMed Scopus (58) Google Scholar, 10Bernhard M. Buhrke T. Bleijlevens B. De Lacey A.L. Fernandez V.M. Albracht S.P. Friedrich B. J. Biol. Chem. 2001; 276: 15592-15597Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 15Brecht M. Van Gastel M. Buhrke T. Friedrich B. Lubitz W. J. Am. Chem. Soc. 2003; 125: 13075-13083Crossref PubMed Scopus (224) Google Scholar). It has been proposed that a non-Fe-S cofactor may be involved in electron transfer instead (10Bernhard M. Buhrke T. Bleijlevens B. De Lacey A.L. Fernandez V.M. Albracht S.P. Friedrich B. J. Biol. Chem. 2001; 276: 15592-15597Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). In this work the double dimeric wild-type RH (RHWT, Fig. 1A) and a derivative denoted as RHstop (Fig. 1B), which forms a monodimer due to mutational truncation of the C terminus of HoxB (14Buhrke T. Lenz O. Porthun A. Friedrich B. Mol. Microbiol. 2004; 51: 1677-1689Crossref PubMed Scopus (56) Google Scholar), were compared. It has been suggested that the RHstop may lack the putative non-Fe-S cofactor (14Buhrke T. Lenz O. Porthun A. Friedrich B. Mol. Microbiol. 2004; 51: 1677-1689Crossref PubMed Scopus (56) Google Scholar). In both preparations, for the first time reduction of Fe-S clusters in the presence of H2 was clearly detected in UV-visible spectra and by XAS at the iron K-edge. Support for reduction of a non-Fe-S cofactor was not obtained. Seemingly, the Fe-S clusters of the RH differ from those of standard hydrogenases. Bacteria Growth and Enzyme Purification—Strains with the initials HF were derived from R. eutropha H16 (DSM428, ATCC 17699). Large scale cultivation of R. eutropha strains, cell harvesting, cell disruption, and preparation of soluble protein extracts were published before (10Bernhard M. Buhrke T. Bleijlevens B. De Lacey A.L. Fernandez V.M. Albracht S.P. Friedrich B. J. Biol. Chem. 2001; 276: 15592-15597Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 14Buhrke T. Lenz O. Porthun A. Friedrich B. Mol. Microbiol. 2004; 51: 1677-1689Crossref PubMed Scopus (56) Google Scholar). RHWT (Fig. 1A) was purified from the RH-overproducing strain R. eutropha HF371(pGE378) as described in Bernhard et al. (10Bernhard M. Buhrke T. Bleijlevens B. De Lacey A.L. Fernandez V.M. Albracht S.P. Friedrich B. J. Biol. Chem. 2001; 276: 15592-15597Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Starting with 50 g of cells (wet weight) yielded 3.7 mg of RH with a specific activity of 1.6 units/mg of protein. The RHstop protein (Fig. 1B) was purified from R. eutropha HF574(pGE567) as a Strep-tag II fusion protein. 2T. Buhrke, O. Lenz, and B. Friedrich, manuscript in preparation. Starting with 18 g of cells yielded 1.5 mg of RHstop with a specific activity of 1.6 units/mg of protein. The homogeneity of the respective protein preparations was investigated by SDS-PAGE analysis and subsequent Coomassie staining. The amount of impurities was quantified by using the Gelscan Professional V5.1 software (BioSciTec, Frankfurt, Germany). After background subtraction, the sum of the percent differential integrated density of the HoxC- and HoxB-specific bands was correlated to the sum of the percent differential integrated density from the contaminating proteins. Assays of Hydrogenase Activity—H2-oxidizing activity was quantified by an amperometric H2 uptake assay as in Pierik et al. (3Pierik A.J. Schmelz M. Lenz O. Friedrich B. Albracht S.P. FEBS Lett. 1998; 438: 231-235Crossref PubMed Scopus (58) Google Scholar) using a H2 electrode with methylene blue as an electron acceptor. One unit of H2 methylene blue oxidoreductase activity was the amount of enzyme that catalyzed the consumption of 1 μmol of H2/min. Protein concentrations were determined according to the protocol of Bradford (18Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (222184) Google Scholar). Analysis of Metal Contents—Atomic absorption spectroscopy (AAS) and total reflection x-ray fluorescence analysis (TRXFA) (19Klockenkämper R. Total Reflection X-ray Fluorescence Analysis. John Wiley & Sons, London, UK1996Google Scholar) were used for quantification of nickel and iron. For AAS, three aliquots of each RH preparation were solubilized overnight in concentrated HNO3 (65%, Suprapur, Merck), then diluted with ultrapure water (Millipore) to 1–5% HNO3 and further diluted for measurements using the transversely heated graphite furnace technique with longitudinal Zeeman-effect background correction on a PerkinElmer Life Sciences Aanalyst 800 spectrometer equipped with an autosampler and WinLab32 software in the laboratory of Dr. Klaus Irrgang (TU-Berlin). TRXFA for simultaneous nickel and iron quantification was performed on a Picotax spectrometer at Röntec (Berlin, Germany) using 1 μl of concentrated and dried protein solutions. Nickel and iron contents were determined against commercial nickel and iron standards (Fluka) in AAS and relative to a gallium standard in TRXFA. Fourier Transform Infrared Spectroscopy (FTIR)—FTIR measurements were carried out with a Bruker IFS66V/S spectrometer equipped with a photovoltaic MCT detector using a resolution of 2 cm-1. The sample compartment was purged with nitrogen. Samples were held in a temperature-controlled (23 °C) gas-tight liquid cell (volume ∼7 μl) with CaF2 windows. FTIR spectra were baseline-corrected using the software available with the spectrometer. EPR Spectroscopy—EPR measurements were performed in the laboratory of Prof. Robert Bittl (FU-Berlin) on an X-band Bruker Elexsys E580 spectrometer equipped with a SHQE resonator and a helium cryostat (Oxford) (microwave frequency of 9.6 GHz). For additional conditions see the figure legends. From each enzyme spectrum in Fig. 2 the background from sample holder and cavity was subtracted. EPR signals in enzyme samples were quantified by comparison with the integrated intensities of signals from CuSO4 solutions used as spin standards (20Albracht S.P.J. van der Linden E. Faber B.W. Biochim. Biophys. Acta. 2003; 1557: 41-49Crossref PubMed Scopus (30) Google Scholar). UV-Visible Spectroscopy—Purified samples of wild-type RH and RHstop were diluted with 20 mm Tris-HCl (pH 8.0) to a protein concentration of 0.64 mg/ml. UV-visible spectra were recorded on a Cary 1E spectrophotometer (Varian) with a spectral resolution of 0.3 nm. To reduce the RH samples, protein solutions were flushed with hydrogen gas for 1 min. Subsequently, the sample was centrifuged (1 min, 12,000 × g) to remove small amounts of precipitated protein. The clear supernatant was immediately re-transferred to the cuvette, and the UV-visible spectrum of the reduced sample was recorded. XAS—X-ray absorption spectra at the iron K-edge were collected at beamline D2 of the EMBL at HASYLAB (DESY, Hamburg, Germany). XAS samples contained 10–20 μl of RH solution (protein concentration 0.4–1 mm). Fluorescence-detected XAS spectra were measured with a 13-element solid-state germanium detector (Canberra) at 20 K as described elsewhere (11Haumann M. Porthun A. Buhrke T. Liebisch P. Meyer-Klaucke W. Friedrich B. Dau H. Biochemistry. 2003; 42: 11004-11015Crossref PubMed Scopus (37) Google Scholar). An absolute energy calibration (accuracy, ±0.1 eV) was performed by monitoring the Bragg reflections of a crystal positioned at the end of the beamline (21Pettifer R.F. Hermes C. J. Appl. Crystallogr. 1985; 18: 404-412Crossref Google Scholar). 3 scans of ∼60-min duration were taken on the same spot (4.5 × 1 mm) of the sample. Comparison of the first and third scan revealed that radiation damage to the samples was absent because the iron K-edge energy remained unchanged. Six scans, obtained on two separate spots of the samples, were averaged for each EXAFS spectrum. Spectra were normalized, and EXAFS oscillations were extracted as in Dau et al. (22Dau H. Liebisch P. Haumann M. Anal. Bioanal. Chem. 2003; 376: 562-583Crossref PubMed Scopus (281) Google Scholar). The energy scale of iron EXAFS spectra was converted to a k-scale using an E0 of 7112 eV; E0 refined to 7120 eV during the EXAFS simulations. Unfiltered k3-weighted spectra were used for least-squares curve-fitting (22Dau H. Liebisch P. Haumann M. Anal. Bioanal. Chem. 2003; 376: 562-583Crossref PubMed Scopus (281) Google Scholar) and for calculation of Fourier transforms representing k-values ranging from 1.85 to 13 Å-1. The data were multiplied by a fractional cosine window (10% at low and high k-side). For EXAFS simulation, complex backscattering amplitudes were calculated using FEFF 7 (23Zabinsky S.I. Rehr J.J. Aukudinov A. Albers R.C. Eller M.J. Phys. Rev. B. Condens. Matter. 1995; 52: 2995-3009Crossref PubMed Scopus (2738) Google Scholar); the amplitude reduction factor S02 was 0.9. For XAS sample preparation, RH solutions were degassed 3 times under vacuum and subsequently flushed for 10 min with H2 gas. H2-reduced samples were filled under argon or H2 atmosphere into Kapton-covered acrylic glass sample holders using syringes previously filled with Ar or H2. The same samples were used for EPR (before and after XAS) and XAS measurements. Comparison of the Ni-Fe Active Site in RHWT and RHstop—To test whether truncation of the C-terminal 55 amino acids of HoxB affected the Ni-Fe site, preparations of RHWT and RHstop were examined by FTIR and EPR measurements. The FTIR spectrum of oxidized RHstop was identical to that of RHWT (Fig. 2A) in showing an intense peak at 1942 cm-1 originating from the Fe-CO and two peaks at 2072 cm-1 and 2081 cm-1 from the two Fe-CN stretching vibrations as previously reported (3Pierik A.J. Schmelz M. Lenz O. Friedrich B. Albracht S.P. FEBS Lett. 1998; 438: 231-235Crossref PubMed Scopus (58) Google Scholar, 10Bernhard M. Buhrke T. Bleijlevens B. De Lacey A.L. Fernandez V.M. Albracht S.P. Friedrich B. J. Biol. Chem. 2001; 276: 15592-15597Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Upon reduction of the samples by H2, the ν(CO) vibration in both spectra shifted to higher frequencies by 18.5 cm-1 (not shown), whereas the ν(CN) vibrations remained unaffected as previously observed for RHWT (10Bernhard M. Buhrke T. Bleijlevens B. De Lacey A.L. Fernandez V.M. Albracht S.P. Friedrich B. J. Biol. Chem. 2001; 276: 15592-15597Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The redox properties of the Ni-Fe active site of RHstop also turned out to be similar to those of RHWT as detected by EPR (Fig. 2B). Flushing of the RH with H2 generates the Ni-C state (Ni(III)-H-) of the Ni-Fe active site with the hydride in a bridging position between nickel and iron (15Brecht M. Van Gastel M. Buhrke T. Friedrich B. Lubitz W. J. Am. Chem. Soc. 2003; 125: 13075-13083Crossref PubMed Scopus (224) Google Scholar). The Ni-C EPR signals (Fig. 2B) were similar in RHWT and RHstop. Signal quantification revealed that greater than 90% of the nickel was converted to Ni(III) in both preparations after incubation with H2 for 10 min, implying similarly effective heterolytic hydrogen cleavage. However, EPR signals from singly reduced Fe-S clusters were completely absent in the Ni-C state. Reduced Fe-S clusters were also not detected when temperatures between 6 and 50 K and microwave power variations between 0.01 and 10 milliwatt were applied (data not shown). In summary, RHWT and RHstop appeared to be similar with respect to the coordination of the iron of the Ni-Fe site both in the oxidized state and in the Ni-C state, which was nearly quantitatively formed in both cases in the presence of hydrogen. Moreover, both preparations showed identical hydrogenase activities (see "Materials and Methods"). The catalytic properties of RHstop were, thus, not affected by the truncation of the C terminus of HoxB. Determination of Metal Contents—Analysis of the nickel contents by AAS in combination with protein determination yielded on the average about 0.6 mol of nickel in the RHWT and close to 1 mol of nickel in the RHstop (Table I). The relatively low nickel content in RHWT can be explained with impurities in the preparations. SDS-PAGE analysis indicated that preparations of RHWT contained sizable amounts of copurified proteins, whereas preparations of RHstop were homogenous (Fig. 3). The amount of impurities in the RHWT sample was quantified by calculating the intensities of HoxC- and HoxB-specific protein bands and the sum of the band intensities derived from the contaminating proteins. According to this analysis the RHWT sample contained about 16% of impurities, whereas the RHstop sample was pure. Therefore, assuming that each HoxBC unit of RHWT also contained one nickel similar to RHstop, it was estimated from the Fe/Ni ratios (Table I), determined by two independent techniques (AAS and TRXFA), that each HoxBC unit of RHWT contained about 8–9 iron atoms, in accordance with previous estimates (10Bernhard M. Buhrke T. Bleijlevens B. De Lacey A.L. Fernandez V.M. Albracht S.P. Friedrich B. J. Biol. Chem. 2001; 276: 15592-15597Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The iron content of RHstop was about 4–6 and, thus, distinctly lower than that of RHWT.Table IMetal contents of RH preparations Ni,Fe/(HoxBC) values represent the average over values from AAS and protein determinations from five independent preparations of both RHWT and RHstop. Mean iron/nickel ratios were derived from the AAS values and from concentrations determined by TRXFA on two preparations of both RHWT and RHstop. The respective error ranges represent S.D.Nickel/[HoxBC]Iron/[HoxBC]Iron/Nickel (AAS, TRXFA)RHWT0.56 ± 0.094.53 ± 0.958.09 ± 1.04, 8.83 ± 1.16RHstop1.03 ± 0.214.83 ± 2.174.69 ± 2.38, 6.12 ± 0.24 Open table in a new tab Based on sequence homologies (see "Discussion") one would expect HoxB to contain three [4Fe-4S] clusters (6Kleihues L. Lenz O. Bernhard M. Buhrke T. Friedrich B. J. Bacteriol. 2000; 182: 2716-2724Crossref PubMed Scopus (116) Google Scholar). Taking also the iron atom of the Ni-Fe site into account, one HoxBC monodimer might contain 13 Fe/Ni. Thus, the experimentally determined iron content of RHWT was significantly lower than expected. Furthermore, HoxBWT seemed to contain more iron than HoxBstop. The double dimeric RHWT may, thus, comprise iron species that are absent in monodimeric RHstop. Detection of Fe-S Cluster Reduction by UV-Visible Spectroscopy—The optical absorption spectra of the oxidized RHWT and RHstop showed, in addition to a peak at 280 nm due to the absorption of the aromatic amino acid residues of the protein, a broad shoulder around 410 nm, presumably due to the presence of Fe-S clusters (Fig. 4, solid lines). That both preparations were fully oxidized was apparent from the absence of any Ni-C EPR signal (data not shown). The extinction coefficients were calculated at 390 and at 420 nm and compared with data from the literature (Table II). The ϵ(420 nm) value determined for RHstop is in good agreement with the value that would be expected in the presence of two cysteinyl-coordinated [2Fe-2S] clusters (49Lippard S.J. Berg J.M. Principles of Bioinorganic Chemistry. University Science Books, Mill Valley, CA1994Google Scholar). The ϵ(420 nm) of RHWT, on the other hand, is significantly higher, confirming the data from the metal analysis which indicate that RHWT contain additional iron species (see "Determination of Metal Contents"). The ϵ(390 nm) values estimated for RHWT as well as for RHstop were significantly larger than the value of ϵ(390 nm) that was expected if only one [4Fe-4S] cluster was present (Table II).Table IIExtinction coefficients of preparations of RHWT and RHstop at 390 and at 420 nm as obtained from the UV-visible spectra (Fig. 4)ϵ(390 nm)ϵ(420 nm)m-1cm-1m-1cm-1RHWToxaThe values for RHWT were corrected by a factor of 1.188 due to the fact that preparations of RHWT contained 16% impurities31,50028,300RHstopox23,90021,800[4Fe-4S]15,200bThese values were taken from Lippard et al. (49)2× [2Fe-2S]19,200bThese values were taken from Lippard et al. (49)a The values for RHWT were corrected by a factor of 1.188 due to the fact that preparations of RHWT contained 16% impuritiesb These values were taken from Lippard et al. (49Lippard S.J. Berg J.M. Principles of Bioinorganic Chemistry. University Science Books, Mill Valley, CA1994Google Scholar) Open table in a new tab When the RH was reduced by flushing of samples with H2, increased absorption was observed in the whole spectral range (not shown); the curvature of the background (proportional to λ-4) suggested its origin from a scattering contribution. Because this background could be removed by centrifugation of H2-flushed samples, apparently a small portion of the RH protein precipitated, thereby light-scattering aggregates were formed. Aggregation was also observed after flushing with argon, which indicated that it was not specifically caused by H2. Reduction of RHWT by flushing with H2 and subsequent removal of aggregates by centrifugation yielded a spectrum with clearly decreased absorption between 350 and 600 nm compared with the oxidized state (Fig. 4A, dotted line); the absorption at ∼280 nm was almost unchanged. Reduction of RHstop with H2 yielded similar results (Fig. 4B, dotted line). The difference spectra (reduced – oxidized RH, insets in Fig. 4, A and B) showed two main minima (bleachings) around 410 and 550 nm, respectively. Whereas bleaching solely around 410 nm has been observed upon reduction of [4Fe-4S] clusters (24Nakamaru-Ogiso E. Yano T. Ohnishi T. Yagi T. J. Biol. Chem. 2002; 277: 1680-1688Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 25Agarwalla S. Stroud R.M. Gaffney B.J. J. Biol. Chem. 2004; 279: 34123-34129Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), two minima as found in the RH spectrum may be attributed to the reduction of [2Fe-2S] clusters (26Pikus J.D. S
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