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245	1	0	_aHeterologous expression and properties of the Q-subunit of the Fe-only hydrogenase from Thermotoga maritima
490	0		_vBiochimica et Biophysica Acta, 1505(2-3), p.209-219, 2001
520	3		_aThermotoga maritima is a hyperthermophilic bacterium that contains a complex, heterotrimeric (KLQ)Fe-only hydrogenase. Sequence analysis indicates that the gene encoding the smallest subunit (Q), hydC, contains a predicted ironsulfur cluster binding motif. However, characterization of the native Q-subunit has been hampered by interference from and the inability to separate intact Q-subunit from the other two subunits (K and L). To investigate the function and properties of the isolated Q-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 Q-subunit and lacks the first 65 amino acid residues. The midpoint potential of the 18 kDa form was 3356 mV at pH 7.0 and 25³C, as measured by direct electrochemistry, and was pH dependent with a pKox of 7.5 and a pKred of 7.7. The oxidized, recombinant Q-subunit was stable at 80³C 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 halflife of approx. 2.5 h. The recombinant Q-subunit was electron transfer competent and was efficiently reduced by pyruvate ferredoxin oxidoreductase from Pyrococcus furiosus, with a Km of 5 WMand a Vmax of 9 U/mg. In contrast, native T. maritima hydrogenase holoenzyme and its separated K-subunit were much less effective electron donors for the Q-subunit, with a Vmax of 0.01 U/mg and 0.1 U/mg, respectively
650	1	4	_aTHERMOTOGA MARITIMA HYDC
650	1	4	_aFE-ONLY HYDROGENASE
650	1	4	_aHETEROLOGOUS GENE EXPRESSION
700	1	2	_aVerhagen, M.F.J.M.
700	1	2	_aO'Rourke, T.W.
700	1	2	_aLal Menon, A.
700	1	2	_aAdams, M.W.W.
856	4	0	_uhttps://drive.google.com/file/d/1yXJ1ll5vIlB212UipC510xT9GpFK4hv5/view?usp=drivesdk _zPara ver el documento ingresa a Google con tu cuenta: @cicy.edu.mx
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