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

作者：Bowman Lisa; Flanagan Lindsey; Fyfe Paul K; Parkin Alison; Hunter William N; Sargent Frank^*

来源：Biochemical Journal, 2014, 458(3): 449-458.

DOI：10.1042/BJ20131520

• hydrogen metabolism
• iron-sulphur cluster [NiFe]-hydrogenase
• oxygen-tolerance
• protein film electrochemistry (PFE)
• Salmonella enterica

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 angstrom and the hydrogenase was observed to comprise associated large and small subunits. The structure indicated that His(229) from the large subunit was close to the proximal [4Fe-3S] cluster in the small subunit. In addition, His(229) was observed to lie close to a buried glutamic acid (Glu(73)), which is conserved in oxygen-tolerant hydrogenases. His(229) and Glu(73) of the Hyd-5 large subunit were found to be important in both hydrogen oxidation activity and the oxygen-tolerance mechanism. Substitution of His(229) or Glu(73) 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 His(229) has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu(73) could play a supporting role in fine-tuning the chemistry of His(229) to enable this function.

• 出版日期2014-3-15