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

TY - JOUR

T1 - How the structure of the large subunit controls function in an oxygen-tolerant [NiFe]-hydrogenase

AU - Bowman, Lisa

AU - Flanagan, Lindsey

AU - Fyfe, Paul K.

AU - Parkin, Alison

AU - Hunter, William N.

AU - Sargent, Frank

PY - 2014/3/15

Y1 - 2014/3/15

N2 - 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 hydrogenase 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, His 229 was observed to lie close to a buried glutamic acid (Glu 73 ), which is conserved in oxygen-tolerant hydrogenases. His 229 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 observedwith oxygen-tolerant [NiFe]-hydrogenases. It is possible thatHis229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73 could play a supporting role in fine-tuning the chemistry of His229 to enable this function.

AB - 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 hydrogenase 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, His 229 was observed to lie close to a buried glutamic acid (Glu 73 ), which is conserved in oxygen-tolerant hydrogenases. His 229 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 observedwith oxygen-tolerant [NiFe]-hydrogenases. It is possible thatHis229 has a role in stabilizing the super-oxidized form of the proximal cluster in the presence of oxygen, and it is proposed that Glu73 could play a supporting role in fine-tuning the chemistry of His229 to enable this function.

KW - Hydrogen metabolism

KW - Iron-sulphur cluster [NiFe]-hydrogenase

KW - Oxygen-tolerance

KW - Protein film electrochemistry (PFE)

KW - Salmonella enterica

UR - http://www.scopus.com/inward/record.url?scp=84896774103&partnerID=8YFLogxK

U2 - 10.1042/BJ20131520

DO - 10.1042/BJ20131520

M3 - Article

C2 - 24428762

AN - SCOPUS:84896774103

SN - 0264-6021

VL - 458

SP - 449

EP - 458

JO - Biochemical journal

JF - Biochemical journal

IS - 3

ER -