On understanding proton transfer to the biocatalytic [Fe-Fe](H) sub-cluster in [Fe-Fe]H(2)ases: QM/MM MD simulations.

Proton transfer to the Fe-Fe sub-cluster in the Desulfovibrio desulfuricans (DdH) and Clostridium pasteurianum (CpI) [Fe-Fe] hydrogenases was investigated by a combination of first principles and empirical molecular dynamics simulations. Pathways that can be inferred from the X-ray crystal structures of DdH and CpI, i.e., (Glu159→Ser198→Glu156→water460→Cys178→DTMA(Fe-Fe) and (Glu282→Ser319→Glu279→water612→Cys299), respectively, were considered. Proton transfer from Cys178 to DTMA in the Fe-Fe sub-cluster in DdH was readily observed in our results, specifically when Fe-Fe was in the reduced state ([Fe(I)-Fe(I)]) or in the mixed valence state for the protonated distal iron Fe(d) (Fe(I)-Fe(II)-H(-)). A concerted mechanism is proposed, where proton transfer in DdH from Glu159 to Glu156 via Ser198 and Glu156 to Cys178 via water460 readily occurred, as well as from Glu282 to Glu279 via Ser319 and Glu279 to Cys299 via water612 in CpI. The theoretical prediction of the proton transfer characteristics is consistent with the assumed biocatalytic mechanism of the [Fe-Fe] hydrogenases in which the proton binds at Fe(d), providing confirmation that has not been explored so far. The computational results were qualitatively validated by the agreement with experimental hydrogen production activity data for mutated CpI enzymes, relative to the wild-type protein. Finally, the insight provided by the simulations, combined, in part, with experimental validation, are important for establishing an approach in future exploration of proton transfer to the active site in this class of enzymes, and possibly also for biomimetic analogs.