tailieunhanh - Báo cáo khoa học: Assignment of the [4Fe-4S] clusters of Ech hydrogenase from Methanosarcina barkeri to individual subunits via the characterization of site-directed mutants

Ech hydrogenase fromMethanosarcina barkeriis a member of a distinct group of membrane-bound [NiFe] hydrogenases with sequence similarity to energy-conserving NADH:quinone oxidoreductase (complex I). The sequence of the enzyme predicts the binding of three [4Fe-4S] clusters, one by subunit EchC and two by subunit EchF. Previous studies had shown that two of these clusters could be fully reduced under 10 5 Pa of H2at pH 7 giving rise to two distinct S½ electron paramagnetic resonance (EPR) signals, designated as the g¼ and theg¼ signal | ềFEBS Journal Assignment of the 4Fe-4S clusters of Ech hydrogenase from Methanosarcina barkeri to individual subunits via the characterization of site-directed mutants Lucia Forzi1 Jurgen Koch1 Adam M. Guss2 Carl G. Radosevich2 William W. Metcalf2 and Reiner Hedderich1 1 Max-Planck-Institute for terrestrialMicrobiology Marburg Germany 2 Department of Microbiology University of Illinois Urbana IL USA Keywords NiFe hydrogenases NADH quinone oxidoreductase complex I Methanogenic archaea iron-sulfur proteins Correspondence R. Hedderich Max-Planck-Institute for terrestrialMicrobiology Karl-von-Frisch Str. D-35043 Marburg Germany Fax 49 6421 178299 Tel 49 6421 178230 E-mail hedderic@ Received 21 June 2005 revised 22 July 2005 accepted 29 July 2005 doi Ech hydrogenase from Methanosarcina barkeri is a member of a distinct group of membrane-bound NiFe hydrogenases with sequence similarity to energy-conserving NADH quinone oxidoreductase complex I . The sequence of the enzyme predicts the binding of three 4Fe-4S clusters one by subunit EchC and two by subunit EchF. Previous studies had shown that two of these clusters could be fully reduced under 105 Pa of H2 at pH 7 giving rise to two distinct S electron paramagnetic resonance EPR signals designated as the g and the g signal. Redox titrations at different pH values demonstrated that these two clusters had a pH-dependent midpoint potential indicating a function in ion pumping. To assign these signals to the subunits of the enzyme a set of M. barkeri mutants was generated in which seven of eight conserved cysteine residues in EchF were individually replaced by serine. EPR spectra recorded from the isolated mutant enzymes revealed a strong reduction or complete loss of the g signal whereas the g signal was still detectable as the major EPR signal in five mutant enzymes. It is concluded that the cluster giving rise to the g signal is the proximal .