Component interactions in the soluble methane monooxygenase system from Methylococcus capsulatus (Bath)
Component interactions in the soluble methane monooxygenase system from Methylococcus capsulatus (Bath)
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DOI：
10.1021/bi990841m
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发表时间：
1999-09-28
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：
Gassner, GT;Lippard, SJ
荚膜甲基球菌（Methylococcus capsulatus）（Bath）的可溶性甲烷单加氧酶系统包括三种蛋白质组分：251-kDa非血红素双核铁羟化酶（MMOH）、39-kDa含铁-硫-和FAD还原酶（MMOR）和16-kDa调节蛋白（MMOB）。通过等温滴定量热法和停流荧光光谱法在3.3至45 ℃的温度范围内测量氧化的MMOH和MMOB或MMOR之间形成复合物的热力学稳定性和动力学。结果，结合从MMOR和MMOB的平衡分析超离心研究的数据，表明游离的MMOR和MMOB作为单体存在于溶液中，并且以2：1的化学计量结合MMOH。通过同时测量氧化酶和羟化酶活性作为稳态周转期间不同蛋白组分浓度的函数，研究了组分相互作用在sMMO催化机制中的作用。sMMO的氧化酶和羟化酶活性的分配高度依赖于MMOR浓度和有机底物的性质。特别地，在超过羟化酶浓度的20%的MMOR浓度下，NADH氧化与甲烷羟化显著地解偶联，但在高达MMOH浓度的MMOR浓度下，NADH氧化与丙烯环氧化保持紧密偶联。稳态动力学数据拟合的模型，包括自由MMOR和MMOH/MMOR复合物的氧化酶活性和羟化酶活性的MMOH/MMOB复合物的数值模拟。这些数据很好地描述了一个模型，其中MMOR和MMOB结合在不同的相互作用位点的羟化酶的非竞争性。MMOB通过以与其对羟化酶的激活和抑制作用一致的方式差异性地加速从MMOR到含有结合底物和产物的MMOH的分子间电子转移来显示其调节作用。
The soluble methane monooxygenase system of Methylococcus capsulatus (Bath) includes three protein components: a 251-kDa non-heme dinuclear iron hydroxylase (MMOH), a 39-kDa iron-sulfur- and FAD-containing reductase (MMOR), and a 16-kDa regulatory protein (MMOB). The thermodynamic stability and kinetics of formation of complexes between oxidized MMOH and MMOB or MMOR were measured by isothermal titration calorimetry and stopped-flow fluorescence spectroscopy at temperatures ranging from 3.3 to 45 degrees C. The results, in conjunction with data from equilibrium analytical ultracentrifugation studies of MMOR and MMOB, indicate that free MMOR and MMOB exist as monomers in solution and bind MMOH with 2:1 stoichiometry. The role of component interactions in the catalytic mechanism of sMMO was investigated through simultaneous measurement of oxidase and hydroxylase activities as a function of varied protein component concentrations during steady-state turnover. The partitioning of oxidase and hydroxylase activities of sMMO is highly dependent on both the MMOR concentration and the nature of the organic substrate. In particular, NADH oxidation is significantly uncoupled from methane hydroxylation at MMOR concentrations exceeding 20% of the hydroxylase concentration but remains tightly coupled to propylene epoxidation at MMOR concentrations ranging up to the MMOH concentration. The steady-state kinetic data were fit to numerical simulations of models that include both the oxidase activities of free MMOR and of MMOH/MMOR complexes and the hydroxylase activity of MMOH/MMOB complexes. The data were well described by a model in which MMOR and MMOB bind noncompetitively at distinct interacting sites on the hydroxylase. MMOB manifests its regulatory effects by differentially accelerating intermolecular electron transfer from MMOR to MMOH containing bound substrate and product in a manner consistent with its activating and inhibitory effects on the hydroxylase.