Acyl and CO Ligands in the [Fe]‐Hydrogenase Cofactor Scramble upon Photolysis

Abstract

Abstract[Fe]‐hydrogenase harbors the iron‐guanylylpyridinol (FeGP) cofactor, in which the Fe(II) complex contains acyl‐carbon, pyridinol‐nitrogen, cysteine‐thiolate and two CO as ligands. Irradiation with UV‐A/blue light decomposes the FeGP cofactor to a 6‐carboxymethyl‐4‐guanylyl‐2‐pyridone (GP) and other components. Previous in vitro biosynthesis experiments indicated that the acyl‐ and CO‐ligands in the FeGP cofactor can scramble, but whether scrambling occurred during biosynthesis or photolysis was unclear. Here, we demonstrate that the [18O1‐carboxy]‐group of GP is incorporated into the FeGP cofactor by in vitro biosynthesis. MS/MS analysis of the 18O‐labeled FeGP cofactor revealed that the produced [18O1]‐acyl group is not exchanged with a CO ligand of the cofactor, indicating that the acyl and CO ligands are scrambled during photolysis rather than biosynthesis, which ruled out any biosynthesis mechanisms allowing acyl/CO ligands scrambling. Time‐resolved infrared spectroscopy indicated that an acyl‐Fe(CO)3 intermediate is formed during photolysis, in which scrambling of the CO and acyl ligands can occur. This finding also suggests that the light‐excited FeGP cofactor has a higher affinity for external CO. These results contribute to our understanding of the biosynthesis and photosensitive properties of this unique H2‐activating natural complex.