Electron-transfer reactions of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath)
Electron-transfer reactions of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath)
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DOI：
10.1021/bi015556t
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发表时间：
2001-12-11
期刊：
影响因子：
2.9
通讯作者：
Lippard, SJ
中科院分区：
文献类型：
--
作者：
Kopp, DA;Gassner, GT;Lippard, SJ
可溶性甲烷单加氧酶（sMMO）催化甲烷被分子氧羟基化生成甲醇和水，这是甲烷氧化菌碳同化的第一步。这种酶包括三种蛋白质组分：含有双核非血红素铁活性位点的羟化酶（MMOH）;促进电子从NADH转移到NMOH的二铁位点的还原酶（MMOR）;和偶联蛋白（NMOB）。MMOR使用非共价结合的FAD辅因子和[2Fe-2S]簇来介导电子转移。从荚膜甲基球菌（Methylococcus capsulatus，Bath）中克隆了MMOR基因，并在大肠杆菌中进行了高效表达。纯化的重组MMOR在所有检查方面，包括活性、质量、辅因子含量和[2Fe-2S]簇的EPR谱，与天然蛋白没有区别。FAD和[2Fe-2S]辅因子的氧化还原电位，在指示剂染料存在下通过还原滴定测定，为FAD（ox/sq），-176 +/- 7 mV; FAD（sq/hq），-266 +/- 15 mV;和[2Fe-2S]（ox/red），-209 +/- 14 mV。MMOR的中点电位不因添加MMOH、MMOB或MMOH和MMOB两者而改变。用停流紫外-可见光谱法研究了MMOR与NADH的反应，并对反应中间体的动力学和光谱性质进行了描述。pH对MMOR氧化还原性质的影响在pH跃变动力学研究中进行了描述和利用，以测量双电子还原MMOR中FAD和[2Fe-2S]辅因子之间电子转移的速率常数为130 +/- 17 s（-1）。确定的热力学和动力学参数显着扩展我们的理解sMMO系统。
Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to afford methanol and water, the first step of carbon assimilation in methanotrophic bacteria. This enzyme comprises three protein components: a hydroxylase (MMOH) that contains a dinuclear nonheme iron active site; a reductase (MMOR) that facilitates electron transfer from NADH to the diiron site of NMOH; and a coupling protein (NMOB). MMOR uses a noncovalently bound FAD cofactor and a [2Fe-2S] cluster to mediate electron transfer. The gene encoding MMOR was cloned from Methylococcus capsulatus (Bath) and expressed in Escherichia coli in high yield. Purified recombinant MMOR was indistinguishable from the native protein in all aspects examined, including activity, mass, cofactor content, and EPR spectrum of the [2Fe-2S] cluster. Redox potentials for the FAD and [2Fe-2S] cofactors, determined by reductive titrations in the presence of indicator dyes, are FAD(ox/sq), - 176 +/- 7 mV; FAD(sq/hq), -266 +/- 15 mV; and [2Fe-2S](ox/red), -209 +/- 14 mV. The midpoint potentials of MMOR are not altered by the addition of MMOH, MMOB, or both MMOH and MMOB. The reaction of MMOR with NADH was investigated by stopped-flow UV-visible spectroscopy, and the kinetic and spectral properties of intermediates are described. The effects of pH on the redox properties of MMOR are described and exploited in pH jump kinetic studies to measure the rate constant of 130 +/- 17 s(-1) for electron transfer between the FAD and [2Fe-2S] cofactors in two-electron-reduced MMOR. The thermodynamic and kinetic parameters determined significantly extend our understanding of the sMMO system.