Importance of the protein framework for catalytic activity of [FeFe]-hydrogenases

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.