Kunz Ryan C, Horng Yih-Chern, Ragsdale Stephen W
Department of Biochemistry, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
J Biol Chem. 2006 Nov 10;281(45):34663-76. doi: 10.1074/jbc.M606715200. Epub 2006 Sep 11.
Methyl-coenzyme M reductase (MCR) catalyzes the final step of methanogenesis in which coenzyme B and methyl-coenzyme M are converted to methane and the heterodisulfide, CoMS-SCoB. MCR also appears to initiate anaerobic methane oxidation (reverse methanogenesis). At the active site of MCR is coenzyme F430, a nickel tetrapyrrole. This paper describes the reaction of the active MCR(red1) state with the potent inhibitor, 3-bromopropanesulfonate (BPS; I50 = 50 nM) by UV-visible and EPR spectroscopy and by steady-state and rapid kinetics. BPS was shown to be an alternative substrate of MCR in an ionic reaction that is coenzyme B-independent and leads to debromination of BPS and formation of a distinct state ("MCR(PS)") with an EPR signal that was assigned to a Ni(III)-propylsulfonate species (Hinderberger, D., Piskorski, R. P., Goenrich, M., Thauer, R. K., Schweiger, A., Harmer, J., and Jaun, B. (2006) Angew. Chem. Int. Ed. Engl. 45, 3602-3607). A similar EPR signal was generated by reacting MCR(red1) with several halogenated sulfonate and carboxylate substrates. In rapid chemical quench experiments, the propylsulfonate ligand was identified by NMR spectroscopy and high performance liquid chromatography as propanesulfonic acid after protonolysis of the MCR(PS) complex. Propanesulfonate formation was also observed in steady-state reactions in the presence of Ti(III) citrate. Reaction of the alkylnickel intermediate with thiols regenerates the active MCR(red1) state and eliminates the propylsulfonate group, presumably as the thioether. MCR(PS) is catalytically competent in both the generation of propanesulfonate and reformation of MCR(red1). These results provide evidence for the intermediacy of an alkylnickel species in the final step in anaerobic methane oxidation and in the initial step of methanogenesis.
甲基辅酶M还原酶（MCR）催化产甲烷作用的最后一步，在此过程中辅酶B和甲基辅酶M被转化为甲烷和异二硫化物CoMS-SCoB。MCR似乎还能启动厌氧甲烷氧化（逆产甲烷作用）。MCR的活性位点是辅酶F430，一种镍四吡咯。本文通过紫外可见光谱和电子顺磁共振光谱以及稳态和快速动力学方法，描述了活性MCR（red1）状态与强效抑制剂3-溴丙烷磺酸盐（BPS；I50 = 50 nM）的反应。结果表明，BPS在一个不依赖辅酶B的离子反应中是MCR的替代底物，该反应导致BPS脱溴并形成一种具有电子顺磁共振信号的独特状态（“MCR（PS）”），该信号被归属于镍（III）-丙磺酸盐物种（欣德贝格尔，D.，皮斯科尔斯基，R.P.，戈恩里希，M.，绍尔，R.K.，施韦格，A.，哈默，J.，和扬，B.（2006年）《德国应用化学》国际版45，3602 - 3607）。通过使MCR（red1）与几种卤代磺酸盐和羧酸盐底物反应，也产生了类似的电子顺磁共振信号。在快速化学淬灭实验中，通过核磁共振光谱和高效液相色谱法确定，在MCR（PS）配合物质子解后，丙磺酸盐配体为丙烷磺酸。在存在柠檬酸钛（III）的稳态反应中也观察到了丙烷磺酸的形成。烷基镍中间体与硫醇的反应使活性MCR（red1）状态再生，并消除丙磺酸盐基团，推测是以硫醚的形式。MCR（PS）在丙烷磺酸的生成和MCR（red1）的重新形成过程中均具有催化活性。这些结果为烷基镍物种在厌氧甲烷氧化的最后一步和产甲烷作用的起始步骤中作为中间体提供了证据。
