Importance of the protein framework for catalytic activity of [FeFe]-hydrogenases |
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Authors: | Knörzer Philipp Silakov Alexey Foster Carina E Armstrong Fraser A Lubitz Wolfgang Happe Thomas |
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Institution: | From the ?AG Photobiotechnologie, Lehrstuhl für Biochemie der Pflanzen, Ruhr-Universität Bochum, 44780 Bochum, Germany.;the §Max-Planck-Institut für Bioanorganische Chemie, 45470 Mülheim an der Ruhr, Germany, and ;the ¶Inorganic Chemistry Laboratory, University of Oxford, Oxford OX1 3QR, United Kingdom |
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Abstract: | The active center (H-cluster) of FeFe]-hydrogenases is embedded into a hydrophobic pocket within the protein. We analyzed several amino acids, located in the vicinity of this niche, by site-directed mutagenesis of the FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA1). These amino acids are highly conserved and predicted to be involved in H-cluster coordination. Characterization of two hydrogenase variants confirmed this hypothesis. The exchange of residues CrHydA1Met(415) and CrHydA1Lys(228) resulted in inactive proteins, which, according to EPR and FTIR analyses, contain no intact H-cluster. However, FeFe]-hydrogenases in which CpIMet(353) (CrHydA1Met(223)) and CpICys(299) (CrHydA1Cys(169)) were exchanged to leucine and serine, respectively, showed a structurally intact H-cluster with catalytic activity either absent (CpIC299S) or strongly diminished (CpIM353L). In the case of CrHydA1C169S, the H-cluster was trapped in an inactive state exhibiting g values and vibrational frequencies that resembled the H(trans) state of DdH from Desulfovibrio desulfuricans. This cysteine residue, interacting with the bridge head nitrogen of the di(methyl)amine ligand, seems therefore to represent an essential contribution of the immediate protein environment to the reaction mechanism. Exchanging methionine CpIM(353) (CrHydA1M(223)) to leucine led to a strong decrease in turnover without affecting the K(m) value of the electron donor. We suggest that this methionine constitutes a "fine-tuning" element of hydrogenase activity. |
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Keywords: | Biophysics Chlamydomonas Electrochemistry Hydrogenase Metalloenzymes Metalloproteins Protein Conformation Protein-Metal Ion Interaction Site-directed Mutagenesis Spectroscopy |
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