Microbial hydrogen splitting in the presence of oxygen.

The origin of the tolerance of a subclass of [NiFe]-hydrogenases to the presence of oxygen was unclear for a long time. Recent spectroscopic studies showed a conserved active site between oxygen-sensitive and oxygen-tolerant hydrogenases, and modifications in the vicinity of the active site in the large subunit could be excluded as the origin of catalytic activity even in the presence of molecular oxygen. A combination of bioinformatics and protein structural modelling revealed an unusual co-ordination motif in the vicinity of the proximal Fe-S cluster in the small subunit. Mutational experiments confirmed the relevance of two additional cysteine residues for the oxygen-tolerance. This new binding motif can be used to classify sequences from [NiFe]-hydrogenases according to their potential oxygen-tolerance. The X-ray structural analysis of the reduced form of the enzyme displayed a new type of [4Fe-3S] cluster co-ordinated by six surrounding cysteine residues in a distorted cubanoid geometry. The unusual electronic structure of the proximal Fe-S cluster can be analysed using the broken-symmetry approach and gave results in agreement with experimental Mößbauer studies. An electronic effect of the proximal Fe-S cluster on the remote active site can be detected and quantified. In the oxygen-tolerant hydrogenases, the hydride occupies an asymmetric binding position in the Ni-C state. This may rationalize the more facile activation and catalytic turnover in this subclass of enzymes.