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Biochem J. 2014 Mar 15;458(3):449-58. doi: 10.1042/BJ20131520.

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

Author information

1
*Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
2
†Department of Chemistry, University of York, Heslington, York YO10 5DD, England, U.K.
3
‡Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, U.K.

Abstract

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.

PMID:
24428762
PMCID:
PMC3940037
DOI:
10.1042/BJ20131520
[Indexed for MEDLINE]
Free PMC Article

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