Heterologous expression and properties of the gamma-subunit of the Fe-only hydrogenase from Thermotoga maritima.
Thermotoga maritima is a hyperthermophilic bacterium that contains a complex, heterotrimeric (alpha(beta)gamma) Fe-only hydrogenase. Sequence analysis indicates that the gene encoding the smallest subunit (gamma), hydC, contains a predicted iron-sulfur cluster binding motif. However, characterization of the native gamma-subunit has been hampered by interference from and the inability to separate intact gamma-subunit from the other two subunits (alpha and beta). To investigate the function and properties of the isolated gamma-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. NH2-terminal sequencing revealed that the 10 kDa protein is a truncated form of the intact gamma-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 degrees C, as measured by direct electrochemistry, and was pH dependent with a pK(ox) of 7.5 and a pK(red) of 7.7. The oxidized, recombinant gamma-subunit was stable at 80 degrees 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 gamma-subunit was electron transfer competent and was efficiently reduced by pyruvate ferredoxin oxidoreductase from Pyrococcus furiosus, with a Km of 5microM and a Vmax of 9 U/mg. In contrast, native T. maritima hydrogenase holoenzyme and its separated alpha-subunit were much less effective electron donors for the gamma-subunit, with a V(max) of 0.01 U/mg and 0.1 U/mg, respectively.
Verhagen
M F
MF
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
O'Rourke
T W
TW
Menon
A L
AL
Adams
M W
MW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D015971:Gene Expression Regulation, Enzymologic; D006864:Hydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D020124:Thermotoga maritima / Q000201:enzymology / Q000235:genetics*
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; C118830:iron hydrogenase; D006864:Hydrogenase