Heterologous expression and properties of the gamma-subunit of the Fe-onlyhydrogenase from Thermotoga maritima

Thermotoga maritima is a hyperthermophilic bacterium that contains a comple x, heterotrimeric (alpha beta gamma) Fe-only hydrogenase. Sequence analysis indicates that the gene encoding the smallest subunit (gamma), hydC, conta ins a predicted iron-sulfur cluster binding motif. However, characterizatio n of the native gamma -subunit has been hampered by interference from and t he inability to separate intact gamma -subunit from the other two subunits (alpha and beta). To investigate the function and properties of the isolate d gamma -subunit, the gene encoding HydG was expressed in Escherichia coli. Two forms of the recombinant protein were obtained with molecular masses o f 10 and 18 kDa, respectively. Both contained a single [2Fe-2S] cluster bas ed on metal analysis, EPR and UV-visible spectroscopy. NH2-terminal sequenc ing revealed that the 10 kDa protein is a truncated form of the intact gamm a -subunit and lacks the first 65 amino acid residues. The midpoint potenti al of the 18 kDa form was -356 mV at pH 7.0 and 25 degreesC, as measured by direct electrochemistry, and was pH dependent with a pK(ox) of 7.5 and a p K(red) of 7.7. The oxidized, recombinant gamma -subunit was stable at 80 de greesC under anaerobic conditions with a half-life greater than 24 h, as ju dged by the UV-visible spectrum of the [2Fe-2S] cluster. In the presence of air the protein was less stable and denatured with a half-life of approx. 2.5 h. The recombinant gamma -subunit was electron transfer competent and w as efficiently reduced by pyruvate ferredoxin oxidoreductase from Pyrococcu s furiosus, with a K-m of 5 muM and a V-max of 9 U/mg. In contrast, native T. maritima hydrogenase holoenzyme and its separated a-subunit were much le ss effective electron donors for the gamma -subunit. with a V-max of 0.01 U /mg and 0.1 U/mg, respectively. (C) 2001 Elsevier Science B.V. All rights r eserved.