Characterization of the iron-sulfur clusters in the nitrogenase-like reductase CfbC/D required for coenzyme F biosynthesis.

Coenzyme F is a nickel-containing tetrapyrrole, serving as the prosthetic group of methyl-coenzyme M reductase in methanogenic and methanotrophic archaea. During coenzyme F biosynthesis, the tetrapyrrole macrocycle is reduced by the nitrogenase-like CfbC/D system consisting of the reductase component CfbC and the catalytic component CfbD. Both components are homodimeric proteins, each carrying a [4Fe-4S] cluster. Here, the ligands of the [4Fe-4S] clusters of CfbC and CfbD were identified revealing an all cysteine ligation of both clusters. Moreover, the midpoint potentials of the [4Fe-4S] clusters were determined to be -256 mV for CfbC and -407 mV for CfbD. These midpoint potentials indicate that the consecutive thermodynamically unfavorable 6 individual "up-hill" electron transfers to the organic moiety of the Ni-sirohydrochlorin a,c-diamide substrate require an intricate interplay of ATP-binding, hydrolysis, protein complex formation and release to drive product formation, which is a common theme in nitrogenase-like systems.