The catalytic mechanism of [FeFe]-hydrogenases, which essentially depends on the dual design concept of protein-scaffold and bioinorganic cofactor, provides the perfect example for an effectively coordinated division of labor and uncovers many of the advantages that biocatalysts have over currently existing catalytic mimics. The active cofactor of [FeFe]-hydrogenases (H-cluster) exhibits a unique FeS-architecture consisting of a classic [4Fe-4S] complex, covalently coupled to a uniquely ligated [2Fe-2S]-moiety, at which H2-turnover occurs. While we have a basic conception of the steps and states of the catalytic cycle, essential aspects still remain as missing pieces of a controversially discussed working hypothesis. Maybe the most painful gap concerns the terminal metal hydride intermediate, which has long been accepted as a central pre-assumption, eponymous for the proposed reversible heterolytic mechanism of H2/H+ conversion. Based on the current model of catalysis, we employed a novel ATR-FTIR-system to selectively enrich and spectroscopically characterize this missing state for three wild-type [FeFe]-hydrogenases and numerous site specific enzyme variants, following Le Chatelier’s principle. We were further able to identify new undescribed states which enable us to unravel the succession and interplay between proton- and electron-transfer steps during catalysis. To understand the communication and mutual influences between cofactor and protein-environment is an essential precondition for being able to introduce the evolutionarily optimized biocatalytic process into the design concept of efficient, robust and sustainable next-generation catalysts. The kinetic, electrochemical and spectroscopic characterization of site-directed enzyme variants targeting both, cofactor and protein, allowed us to assign essential individual functionalities to cofactor parts and polypeptide positions in the secondary ligand sphere of the cofactor, in the redox-partner binding site and the proton-transfer pathway. It furthermore provides important insights into the process of oxygen-dependent H-cluster degradation, which is the most relevant obstacle for establishing sustainable industrial applications that exploit the high catalytic efficiency and moderate turnover conditions of [FeFe]-hydrogenases.

Probing transient states in the catalytic cycle of [FeFe]-hydrogenases

254th National Meeting (2017)

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