Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, United States.
Biochemistry. 2010 Dec 28;49(51):10902-11. doi: 10.1021/bi101562m. Epub 2010 Dec 2.
Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the terminal step in methanogenesis using coenzyme B (CoBSH) as the two-electron donor to reduce methyl-coenzyme M (methyl-SCoM) to form methane and the heterodisulfide, CoBS-SCoM. The active site of MCR contains an essential redox-active nickel tetrapyrrole cofactor, coenzyme F(430), which is active in the Ni(I) state (MCR(red1)). Several catalytic mechanisms have been proposed for methane synthesis that mainly differ in whether an organometallic methyl-Ni(III) or a methyl radical is the first catalytic intermediate. A mechanism was recently proposed in which methyl-Ni(III) undergoes homolysis to generate a methyl radical (Li, X., Telser, J., Kunz, R. C., Hoffman, B. M., Gerfen, G., and Ragsdale, S. W. (2010) Biochemistry 49, 6866-6876). Discrimination among these mechanisms requires identification of the proposed intermediates, none of which have been observed with native substrates. Apparently, intermediates form and decay too rapidly to accumulate to detectible amounts during the reaction between methyl-SCoM and CoBSH. Here, we describe the reaction of methyl-SCoM with a substrate analogue (CoB(6)SH) in which the seven-carbon heptanoyl moiety of CoBSH has been replaced with a hexanoyl group. When MCR(red1) is reacted with methyl-SCoM and CoB(6)SH, methanogenesis occurs 1000-fold more slowly than with CoBSH. By transient kinetic methods, we observe decay of the active Ni(I) state coupled to formation and subsequent decay of alkyl-Ni(III) and organic radical intermediates at catalytically competent rates. The kinetic data also revealed substrate-triggered conformational changes in active Ni(I)-MCR(red1). Electron paramagnetic resonance (EPR) studies coupled with isotope labeling experiments demonstrate that the radical intermediate is not tyrosine-based. These observations provide support for a mechanism for MCR that involves methyl-Ni(III) and an organic radical as catalytic intermediates. Thus, the present study provides important mechanistic insights into the mechanism of this key enzyme that is central to biological methane formation.
甲基辅酶 M 还原酶（MCR）来源于产甲烷古菌，利用辅酶 B（CoBSH）作为两电子供体，催化产甲烷作用的最后一步，将甲基辅酶 M（methyl-SCoM）还原为甲烷和异双硫物 CoBS-SCoM。MCR 的活性部位含有一个必需的氧化还原活性镍四吡咯辅因子，辅酶 F（430），它在 Ni（I）状态下具有活性（MCR（red1））。已经提出了几种甲烷合成的催化机制，主要区别在于第一个催化中间物是有机金属甲基-Ni（III）还是甲基自由基。最近提出了一种机制，其中甲基-Ni（III）经历均裂生成甲基自由基（Li，X.，Telser，J.，Kunz，R. C.，Hoffman，B. M.，Gerfen，G.，和 Ragsdale，S. W.（2010）生物化学 49，6866-6876）。这些机制之间的区分需要鉴定所提出的中间物，而这些中间物都没有在天然底物中观察到。显然，中间物形成和衰减太快，以至于在甲基-SCoM 和 CoBSH 之间的反应过程中无法积累到可检测的量。在这里，我们描述了甲基-SCoM 与底物类似物（CoB（6）SH）的反应，其中 CoBSH 的七碳庚酰部分被己酰基取代。当 MCR（red1）与甲基-SCoM 和 CoB（6）SH 反应时，与 CoBSH 相比，甲烷生成的速度慢 1000 倍。通过瞬态动力学方法，我们观察到活性 Ni（I）状态的衰减与烷基-Ni（III）和有机自由基中间物的形成和随后的衰减以催化能力的速率耦合。动力学数据还揭示了活性 Ni（I）-MCR（red1）中的底物触发构象变化。电子顺磁共振（EPR）研究与同位素标记实验相结合，证明自由基中间物不是基于酪氨酸的。这些观察结果为 MCR 的一种机制提供了支持，该机制涉及甲基-Ni（III）和有机自由基作为催化中间物。因此，本研究为该关键酶的机制提供了重要的机制见解，该酶是生物甲烷形成的核心。
