[Fe]-hydrogenase: biosynthesis of the FeGP cofactor and its catalytic function

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The iron-guanylylpyridinol (FeGP) cofactor is the prosthetic group of [Fe]-hydrogenase, which catalyzes reversible hydride transfer from H2 to methenyl-tetrahydromethanopterin. This reaction is involved in the hydrogenotrophic methanogenic pathway. In the FeGP cofactor, a low-spin Fe(II) is fixed by pyridinol-N and acyl-C from the pyridinol moiety, and the iron is coordinated with two CO ligands and one cysteine-S. Mutation analysis has indicated that the Cys176-S-Fe bonding is crucial for the activity of [Fe]-hydrogenase. This cofactor is biosynthesized by the reactions catalyzed by HcgA-G proteins. HcgA and HcgG are iron-sulfur cluster containing enzymes. We have previously identified the function of HcgB, HcgE and HcgF based on structural and biochemical analyses. We also proposed HcgD as an iron-trafficking protein for insertion of iron into the cofactor precursor. In the first half of this Priority program, we identified that the S-adenosyl-methionine-dependent methyltransferase-reaction of HcgC uses chemically synthesized pyridinol precursor and unveiled the unique catalytic mechanism of HcgC. Based on this finding, we established the methods for preparation of the guanylylpyridinol precursor from chemically synthesized pyridinol precursor using HcgB and HcgC. We also found a novel enzyme activity, which can repair the partially light-decomposed FeGP cofactor. In addition, we solved the crystal structure of [Fe]-hydrogenase in the closed active conformation at an atomic resolution (1.06 Å), in which the precise structure of the FeGP cofactor in the activated enzyme was indicated. Based on the active form structure, we proposed the catalytic functions of the FeGP cofactor. In the second half of this program, we would like to study further biosynthesis of the FeGP cofactor; we will use the in vitro biosynthesis methods using the chemically synthesized pyridinol precursor as the substrate. We would like to also characterize the enzyme activity of each Hcg protein using biochemical techniques and delta hcg mutants. We will study the enzyme activity that repairs the light-decomposed FeGP cofactor. As the second fundamental area expected for this Priority program, we will study the function of the FeGP cofactor in the catalytic mechanism of [Fe]-hydrogenase. For this aim, we will use several spectroscopic methods (infrared, Mössbauer, resonance Raman and Nuclear resonance vibrational spectroscopy) and density function theory calculation in collaboration with the groups in this Priority program.