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. In the present study, 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 hydro-genase was observed to comprise associated large and small subunits. The structure indicated that His²²⁹ from the large subunit was close to the proximal [4Fe-3S] cluster in the small subunit. In addition, His²²⁹ was observed to lie close to a buried glutamic acid (Glu⁷³), which is conserved in oxygen-tolerant hydrogenases. His²²⁹ and Glu⁷³ of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His²²⁹ or Glu⁷³ with alanine led to a loss in the ability of Hyd-5 to oxidize 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 His²²⁹ has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu⁷³could play a supporting role in fine-tuning the chemistry of His²²⁹ to enable this function.