Biosynthesis of the Iron-Guanylylpyridinol Cofactor of [Fe]-Hydrogenase in Methanogenic Archaea as Elucidated by Stable-Isotope Labeling

[Fe]-hydrogenase catalyzes the reversible hydride transfer from H₂ to methenyltetrahydromethanoptherin, which is an intermediate in methane formation from H₂ and CO₂ in methanogenic archaea. The enzyme harbors a unique active site iron-guanylylpyridinol (FeGP) cofactor, in which a low-spin Fe^II is coordinated by a pyridinol-N, an acyl group, two carbon monoxide, and the sulfur of the enzyme’s cysteine. Here, we studied the biosynthesis of the FeGP cofactor by following the incorporation of ¹³C and ²H from labeled precursors into the cofactor in growing methanogenic archaea and by subsequent NMR, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) and IR analysis of the isolated cofactor and reference compounds. The pyridinol moiety of the cofactor was found to be synthesized from three C-1 of acetate, two C-2 of acetate, two C-1 of pyruvate, one carbon from the methyl group of l-methionine, and one carbon directly from CO₂. The metabolic origin of the two CO-ligands was CO₂ rather than C-1 or C-2 of acetate or pyruvate excluding that the two CO are derived from dehydroglycine as has previously been shown for the CO-ligands in [FeFe]-hydrogenases. A formation of CO from CO₂ via direct reduction catalyzed by a nickel-dependent CO dehydrogenase or from formate could also be excluded. When the cells were grown in the presence of ¹³CO, the two CO-ligands and the acyl group became ¹³C-labeled, indicating either that free CO is an intermediate in their synthesis or that free CO can exchange with these iron-bound ligands. Based on these findings, we propose pathways for how the FeGP cofactor might be synthesized.