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首页> 外文期刊>Biochemistry >Detection of Organometallic and Radical Intermediates in the Catalytic Mechanism of Methyl-Coenzyme M Reductase Using the Natural Substrate Methyl-Coenzyme M and a Coenzyme B Substrate Analogue
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Detection of Organometallic and Radical Intermediates in the Catalytic Mechanism of Methyl-Coenzyme M Reductase Using the Natural Substrate Methyl-Coenzyme M and a Coenzyme B Substrate Analogue

机译：使用天然底物甲基辅酶M和辅酶B底物类似物检测甲基辅酶M还原酶催化机理中的有机金属和自由基中间体

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摘要

Methyl-coenzyme M reductase (MCR) from methanogenic archaea catalyzes the terminal stepnin methanogenesis using coenzyme B (CoBSH) as the two-electron donor to reduce methyl-coenzymenM(methyl-SCoM) to form methane and the heterodisulfide, CoBS-SCoM. The active site ofMCR contains annessential redox-active nickel tetrapyrrole cofactor, coenzyme F430, which is active in theNi(I) state (MCRred1).nSeveral catalytic mechanisms have been proposed for methane synthesis that mainly differ in whether annorganometallicmethyl-Ni(III) or amethyl radical is the first catalytic intermediate.Amechanismwas recentlynproposed in which methyl-Ni(III) undergoes homolysis to generate a methyl radical (Li, X., Telser, J., Kunz,nR. C., Hoffman, B.M., Gerfen, G., and Ragsdale, S.W. (2010) Biochemistry 49, 6866-6876). Discriminationnamong these mechanisms requires identification of the proposed intermediates, none of which have beennobserved with native substrates. Apparently, intermediates form and decay too rapidly to accumulate tondetectible amounts during the reaction between methyl-SCoMand CoBSH. Here, we describe the reaction ofnmethyl-SCoM with a substrate analogue (CoB6SH) in which the seven-carbon heptanoyl moiety of CoBSHnhas been replaced with a hexanoyl group.WhenMCRred1 is reacted with methyl-SCoMand CoB6SH, meth-nanogenesis occurs 1000-foldmore slowly than with CoBSH. By transient kineticmethods,we observe decay ofnthe active Ni(I) state coupled to formation and subsequent decay of alkyl-Ni(III) and organic radical inter-nmediates at catalytically competent rates. The kinetic data also revealed substrate-triggered conformationalnchanges in active Ni(I)-MCRred1. Electron paramagnetic resonance (EPR) studies coupled with isotope labelingnexperiments demonstrate that the radical intermediate is not tyrosine-based. These observations providensupport for amechanismforMCR that involvesmethyl-Ni(III) and an organic radical as catalytic intermediates.nThus, the present study provides importantmechanistic insights into themechanismof this key enzyme that isncentral to biological methane formation.
机译：产甲烷古菌中的甲基辅酶M还原酶（MCR）使用辅酶B（CoBSH）作为双电子供体，催化末端步骤的产甲烷菌甲烷化反应，以还原甲基辅酶M（methyl-SCoM）形成甲烷和杂二硫键CoBS-SCoM。 MCR的活性位点包含非活性的氧​​化还原活性四吡咯镍辅因子F430，其在Ni（I）状态下具有活性（MCRred1）.n已提出了甲烷合成的几种催化机制，主要区别在于有机金属甲基-Ni（III）还是甲烷。近年来，人们提出了一种机制，其中对甲基镍（III）进行均解以生成一个甲基自由基（Li，X.，Telser，J.，Kunz，nR.C。，Hoffman，BM，Gerfen，G和Ragsdale，SW（2010）Biochemistry 49，6866-6876）。在这些机制之间的区别需要鉴定所提出的中间体，而这些中间体都没有天然底物。显然，中间体在甲基-SCoM和CoBSH之间的反应过程中形成和分解的速度过快，无法积累大量可检测的量。在这里，我们描述了n甲基-SCoM与底物类似物（CoB6SH）的反应，其中CoBSHn的7个碳的庚酰基部分已被己酰基取代。当MCRred1与甲基-SCoM和CoB6SH反应时，甲基纳米发生的速度要慢1000倍比使用CoBSH。通过瞬态动力学方法，我们观察到活性Ni（I）态的衰减与形成的催化态以及随后的烷基-Ni（III）和有机自由基的中间衰减以催化有效速率相关。动力学数据还揭示了活性Ni（I）-MCRred1中底物触发的构象变化。电子顺磁共振（EPR）研究与同位素标记实验相结合表明，自由基中间体不是基于酪氨酸的。这些观察结果为涉及甲基-Ni（III）和有机自由基作为催化中间体的MCR机理提供了支持。因此，本研究为这种关键酶的生物机理提供了重要的力学见解，而这种关键酶与生物甲烷的形成无关。

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  • 来源
    《Biochemistry》 |2010年第51期|p.10902-10911|共10页
  • 作者

    Mishtu Dey Xianghui Li Ryan C. Kunz and Stephen W. Ragsdale;

  • 作者单位

    Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.‡Current address: Department of Chemistry, Massachusetts Institute of Technology, Cambridge,Massachusetts 02139.§Current address: New England Biolabs, Inc., Shanghai R&D Center, Shanghai, China.) Current address: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115;

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