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

Lisa Bowman,William N Hunter,Frank Sargent,Alison Parkin,Lindsey Flanagan,Paul K Fyfe

doi:10.1042/bj20131520

Lisa Bowman, William N Hunter + Show 4 more

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https://doi.org/10.1042/bj20131520

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Journal: Biochemical Journal	Publication Date: Feb 28, 2014
Citations: 36	License type: CC BY 3.0

Affiliation: University of Dundee, University of York

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.

Highlights

Salmonella enterica serovar Typhimurium is a Gram-negative γ -Proteobacterium and an opportunistic animal pathogen
Two of these encode homologues of the Hyd-1 and Hyd-2 [NiFe]hydrogenase enzymes found in Escherichia coli [6,7,8] and in the present study the E. coli nomenclature will be applied to these S. enterica enzymes
The LB03 (PT5, hydA ) transmembrane domain (TM) − His strain was cultured at a small scale under anoxic conditions and whole cells were assayed for Benzyl Viologen (BV)-linked hydrogenase activity (Figure 2A)

Summary

Introduction

Salmonella enterica serovar Typhimurium is a Gram-negative γ -Proteobacterium and an opportunistic animal pathogen. H2 oxidation (or ‘uptake’) is catalysed in S. enterica by three [NiFe]-hydrogenases encoded by the hyaABCDEF (STM1786– STM1791), hybOABCEDFG (STM3150–STM3143), and hydABCDEFGHI (STM1539–STM1531) operons. Two of these (hya and hyb) encode homologues of the Hyd-1 and Hyd-2 [NiFe]hydrogenase enzymes found in Escherichia coli [6,7,8] and in the present study the E. coli nomenclature will be applied to these S. enterica enzymes. All three S. enterica H2-uptake enzymes comprise similar ‘core’ structures with a large subunit (or α-subunit) containing the [Ni–Fe–CO–2CN− ] catalytic centre, and a small subunit (or β-subunit) containing three Fe–S clusters. The three S. enterica H2-uptake enzymes can be subcategorized as ‘membrane-bound’ [NiFe]-hydrogenases

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