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Maturation of FeFe hydrogenases requires the biosynthesis and insertion of the catalytic iron-sulfur cluster, the H cluster. Two radical S-adenosylmethionine (SAM) proteins proposed to function in H cluster biosynthesis, HydEF and HydG, were recently identified in the hydEF-1 mutant of the green alga Chlamydomonas reinhardtii (M. C. Posewitz, P. W. King, S. L. Smolinski, L. Zhang, M. Seibert, and M. L. Ghirardi, J. Biol. Chem. 279:25711-25720, 2004). Previous efforts to study FeFe hydrogenase maturation in Escherichia coli by coexpression of C. reinhardtii HydEF and HydG and the HydA1 FeFe hydrogenase were hindered by instability of the hydEF and hydG expression clones. A more stable FeFe hydrogenase expression system has been achieved in E. coli by cloning and coexpression of hydE, hydF, and hydG from the bacterium Clostridium acetobutylicum. Coexpression of the C. acetobutylicum maturation proteins with various algal and bacterial FeFe hydrogenases in E. coli resulted in purified enzymes with specific activities that were similar to those of the enzymes purified from native sources. In the case of structurally complex FeFe hydrogenases, maturation of the catalytic sites could occur in the absence of an accessory iron-sulfur cluster domain. Initial investigations of the structure and function of the maturation proteins HydE, HydF, and HydG showed that the highly conserved radical-SAM domains of both HydE and HydG and the GTPase domain of HydF were essential for achieving biosynthesis of active FeFe hydrogenases. Together, these results demonstrate that the catalytic domain and a functionally complete set of Hyd maturation proteins are fundamental to achieving biosynthesis of catalytic FeFe hydrogenases.
King et al. (Thu,) studied this question.