Heterologous expression and properties of the γ-subunit of the Fe-only hydrogenase from Thermotoga maritima

Abstract

Thermotoga maritima is a hyperthermophilic bacterium that contains a complex, heterotrimeric (αβγ) Fe-only hydrogenase. Sequence analysis indicates that the gene encoding the smallest subunit (γ), hydC, contains a predicted iron-sulfur cluster binding motif. However, characterization of the native γ-subunit has been hampered by interference from and the inability to separate intact γ-subunit from the other two subunits (α and β). To investigate the function and properties of the isolated γ-subunit, the gene encoding HydG was expressed in Escherichia coli. Two forms of the recombinant protein were obtained with molecular masses of 10 and 18 kDa, respectively. Both contained a single [2Fe-2S] cluster based on metal analysis, EPR and UV-visible spectroscopy. NH 2-terminal sequencing revealed that the 10 kDa protein is a truncated form of the intact γ-subunit and lacks the first 65 amino acid residues. The midpoint potential of the 18 kDa form was −356 mV at pH 7.0 and 25°C, as measured by direct electrochemistry, and was pH dependent with a p K ox of 7.5 and a p K red of 7.7. The oxidized, recombinant γ-subunit was stable at 80°C under anaerobic conditions with a half-life greater than 24 h, as judged 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 γ-subunit was electron transfer competent and was efficiently reduced by pyruvate ferredoxin oxidoreductase from Pyrococcus furiosus, with a K m of 5 μM and a V max of 9 U/mg. In contrast, native T. maritima hydrogenase holoenzyme and its separated α-subunit were much less effective electron donors for the γ-subunit, with a V max of 0.01 U/mg and 0.1 U/mg, respectively.