Importance of the Protein Framework for Catalytic Activity of [FeFe]-Hydrogenases

Philipp Knörzer; Alexey Silakov; Carina E. Foster; Fraser A. Armstrong; Wolfgang Lubitz; Thomas Happe

doi:10.1074/jbc.M111.305797

Importance of the Protein Framework for Catalytic Activity of [FeFe]-Hydrogenases

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

↵1 Supported by the EU-SOLAR-H2 program, the Bundesministerium für Bildung und Forschung (BMBF) (Bio-H2), and the Volkswagen (VW) foundation (LigH2t). To whom correspondence should be addressed: Ruhr Universität Bochum, Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie, Univesitätsstrasse 150, 44780 Bochum, Germany. Tel.: 49-234-32-27026; Fax: 49-234-32-14322; E-mail: thomas.happe{at}rub.de.

Background: Hydrogenases contain a unique oxygen-labile metal cofactor.

Results: Substitution of noncovalently interacting residues degrades the catalytic cofactor (K358N and M497L) or reduces activity but leaves the cofactor chemically intact (C299S and M353L).

Conclusion: Lys³⁵⁸ and Met⁴⁹⁷ are essential for H-cluster coordination. Cys²⁹⁹ and Met³⁵³ influence catalytic activity only.

Significance: Understanding specific cofactor-amino acid interactions provides an important basis for improving artificial hydrogen catalysts.

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 CrHydA1Met⁴¹⁵ and CrHydA1Lys²²⁸ resulted in inactive proteins, which, according to EPR and FTIR analyses, contain no intact H-cluster. However, [FeFe]-hydrogenases in which CpIMet³⁵³ (CrHydA1Met²²³) and CpICys²⁹⁹ (CrHydA1Cys¹⁶⁹) 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 CpIM³⁵³ (CrHydA1M²²³) 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.