In Vitro Biosynthesis of the [Fe]‐Hydrogenase Cofactor Verifies the Proposed Biosynthetic Precursors

In the FeGP cofactor of [Fe]‐hydrogenase, low‐spin FeII is in complex with two CO ligands and a pyridinol derivative; the latter ligates the iron with a 6‐acylmethyl substituent and the pyridinol nitrogen. A guanylylpyridinol derivative, 6‐carboxymethyl‐3,5‐dimethyl‐4‐guanylyl‐2‐pyridinol (3), is produced by the decomposition of the FeGP cofactor under irradiation with UV‐A/blue light and is also postulated to be a precursor of FeGP cofactor biosynthesis. HcgC and HcgB catalyze consecutive biosynthesis steps leading to 3. Here, we report an in vitro biosynthesis assay of the FeGP cofactor using the cell extract of the ΔhcgBΔhcgC strain of Methanococcus maripaludis, which does not biosynthesize 3. We chemically synthesized pyridinol precursors 1 and 2, and detected the production of the FeGP cofactor from 1, 2 and 3. These results indicated that 1, 2 and 3 are the precursors of the FeGP cofactor, and the carboxy group of 3 is converted to the acyl ligand. The FeGP cofactor is the prosthetic group of [Fe]‐hydrogenase. The in vitro biosynthesis of this cofactor from synthesized precursors using the cell extract of a mutated strain of Methanococcus maripaludis provided evidence for the biosynthesis of the FeGP cofactor from 6‐carboxymethyl‐2‐pyridinols, an organic CO donor, and/or CO and other substances. This method paves the way to analyze the biosynthetic machinery of this H2‐activation cofactor.