How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase

Salmonella enterica is an opportunistic pathogen that produces a [NiFe]-hydrogenase under aerobic conditions. Here, genetic engineering approaches were used to facilitate isolation of this enzyme, termed Hyd-5. The crystal structure was determined to a resolution of 3.2 Å and the hydrogenase was observed to comprise associated large and small subunits. The structure indicated that residue H229 from the large subunit was close to the proximal [4Fe-3S] cluster in the small subunit. In addition, H229 was observed to lie close to a buried glutamic acid (E73), which is conserved in oxygen-tolerant hydrogenases. Residues H229 and E73 of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen tolerance mechanism. Substitution of H229 or E73 with alanine led to a loss in the ability of Hyd-5 to oxidise hydrogen in air. Furthermore, the H229A variant was found to have lost the overpotential requirement for activity that is always observed with oxygen-tolerant [NiFe]-hydrogenases. It is possible that H229 has a role in stabilising the super-oxidised form of the proximal cluster in the presence of oxygen, and it is proposed that E73 could play a supporting role in fine-tuning the chemistry of H229 to enable this function.
Source: BJ Energy - Category: Biochemistry Authors: Tags: BJ Biomolecules Source Type: research