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

铁-鸟苷酸-吡啶醇辅基(FeGP)是[Fe]-氢酶的辅基，催化氢化物从H2向亚甲基四氢甲烷蝶呤的可逆转移。这一反应参与了氢遗传营养产甲烷途径。在FeGP辅因子中，一个低自旋的Fe(II)被来自吡啶酚部分的吡啶酚-N和酰基-C固定，铁与两个CO配体和一个半胱氨酸-S配位。突变分析表明，Cys176-S-Fe键对[Fe]-氢酶的活性至关重要。这种辅因子是由HcgA-G蛋白催化的反应生物合成的。HcgA和HcgG是含铁硫簇的酶。我们之前已经根据结构和生化分析确定了HcgB、HcgE和HcgF的功能。我们还建议HcgD作为一种铁运输蛋白，将铁插入辅因子前体中。在这个优先计划的前半部分，我们确认了HCGC的S-腺苷-蛋氨酸依赖的甲基转移酶反应使用了化学合成的吡啶醇前体，并揭示了HCGC独特的催化机理。基于这一发现，我们建立了用HcgB和HCGC从化学合成的吡啶酚前驱体制备鸟苷酸吡啶醇前驱体的方法。我们还发现了一种新的酶活性，它可以修复部分光解的FeGP辅因子。此外，我们还在原子分辨率(1.06？)下解决了[Fe]-氢酶在闭合活性构象中的晶体结构，揭示了激活酶中FeGP辅因子的精确结构。根据FeGP辅因子的活性结构，提出了FeGP辅因子的催化功能。在本计划的后半部分，我们将进一步研究FeGP辅因子的生物合成；我们将使用以化学合成的吡啶酚前体为底物的体外生物合成方法。我们还想利用生化技术和增量hcg突变体来表征每种hcg蛋白的酶活性。我们将研究修复光解FeGP辅因子的酶活性。作为这一优先计划的第二个基本领域，我们将研究FeGP辅因子在[Fe]-氢酶催化机制中的作用。为此，我们将使用几种光谱方法(红外、穆斯堡尔、共振拉曼和核共振振动光谱)和密度泛函理论计算，与本优先计划中的小组合作。

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.