MOLECULAR RECOGNITION IN CYTOCHROME-P-450 - MECHANISM FOR THE CONTROL OF UNCOUPLING REACTIONS
MOLECULAR RECOGNITION IN CYTOCHROME-P-450 - MECHANISM FOR THE CONTROL OF UNCOUPLING REACTIONS
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DOI：
10.1021/bi00094a009
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发表时间：
1993-11-02
期刊：
影响因子：
2.9
通讯作者：
SLIGAR, SG
中科院分区：
文献类型：
--
作者：
LOIDA, PJ;SLIGAR, SG
在细胞色素P-450单加氧酶系统中利用吡啶核苷酸衍生的还原当量的途径有三个主要的分支点。第一个是亚铁氧化血红素加合物的自氧化和单加氧酶化学计量所需的第二还原当量的输入之间的分配。第二个是在双氧键断裂和释放双电子还原的O2作为过氧化氢之间。第三个是由一个假定的高价铁氧物种和减少这个中间体由两个额外的电子，以产生水的整体氧化酶化学计量之间的基板氢提取。对于所有调查的基板，在第一个分支点的直接释放的超氧化物从来没有竞争与第二个电子输入。为了阐明底物-P-450复合物的分子识别方面，影响催化循环中的这些单独的分支点，我们测量了在羟基化底物、过氧化氢和水中回收的NADH衍生的还原当量，用于一系列活性位点突变体，其设计用于改变β-羟基化的偶联。我们发现，在第二和第三个分支点的反应特异性是受定点突变，改变的结合口袋的拓扑结构。所有突变体观察到的增加的承诺催化表明，活性位点水合作用是重要的解偶联，形成过氧化氢在第二个分支点。过氧化氢的释放与口袋中突变的位置不相关，如所预期的，如果双电子还原的双氧结合的中间体不直接参与底物活化步骤。然而，在第三分支点处的水产生与底物结合位点中的氨基酸侧链的位置和大小之间观察到强相关性。在结合口袋的上部区域中引入的较大疏水侧链使羟基化产物与水产生的比率相对于野生型增加2-4倍，而在血红素平面附近的残基中的类似取代导致产物减少。总体而言，羟基化和氧化酶活性之间的分区变化超过65%，由于非极性取代基的位置工程到活性位点。底物进入血红素是在推定的铁氧物种水平上紧密耦合的关键因素。这些结果进一步证明了含有单个氧原子的离散中间体（例如，铁氧络合物[FeO]3+）是底物羟基化和输入两个额外的电子当量以形成水的前体。
The pathway for utilization of pyridine nucleotide derived reducing equivalents in the cytochrome P-450 monooxygenase systems has three major branch points. The first is a partitioning between autoxidation of a ferrous, oxygenated heme adduct and input of the second reducing equivalent required for monooxygenase stoichiometry. The second is between dioxygen bond scission and release of two-electron-reduced O2 as hydrogen peroxide. The third is between substrate hydrogen abstraction initiated by a putative higher valent iron-oxo species and reduction of this intermediate by two additional electrons to produce water in an overall oxidase stoichiometry. For all substrates investigated, the direct release of superoxide at the first branch point never competes with second electron input. In order to elucidate the aspects of molecular recognition of a substrate-P-450 complex which affect these individual branch points in the catalytic cycle, we have measured the NADH-derived reducing equivalents recovered in hydroxylated substrate, hydrogen peroxide, and water for a series of active-site mutants designed to alter the coupling of ethylbenzene hydroxylation. We find that the reaction specificity at the second and third branch points is affected by site-directed mutations that alter the topology of the binding pocket. The increased commitment to catalysis observed for all mutants suggests that active-site hydration is important in the uncoupling to form hydrogen peroxide at the second branch point. The liberation of hydrogen peroxide does not correlate with the location of the mutation in the pocket, as expected if the two-electron-reduced dioxygen-bound intermediate is not directly participating in the substrate activation step. However, a strong correlation is observed between water production at the third branch point and the location and size of the amino acid side chain in the substrate binding site. Larger hydrophobic side chains introduced in the upper regions of the binding pocket increase the ratio of hydroxylated product to water production by 2-4-fold relative to wild-type, while similar substitutions in residues near the heme plane result in diminished product. Overall, the partitioning between hydroxylation and oxidase activities varies by over 65% due to the location of nonpolar substituents engineered into the active site. Substrate access to the heme is the key factor for tight coupling at the level of the putative iron-oxo species. These results are further evidence that a discrete intermediate containing a single oxygen atom (e.g., a ferryl-oxo complex, [FeO]3+) is the precursor to both substrate hydroxylation and the input of two additional electron equivalents to form water.