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首页> 外文期刊>Biochemistry >Replacement of the Axial Histidine Ligand with imidazole in Cytochrome c Peroxidase.2.Effects on Heme Coordination and Function
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Replacement of the Axial Histidine Ligand with imidazole in Cytochrome c Peroxidase.2.Effects on Heme Coordination and Function

机译：在细胞色素c过氧化物酶中用咪唑取代轴向组氨酸配体。2．对血红素配位和功能的影响

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The inability of imidazole to complement function in the axial histidine deletion mutant, H175G, of yeast cytochrome c peroxidase has been an intriguing but unresolved issue that impacts our understanding of the role of axial ligands in heme catalysis. Here we report the functional and spectroscopic properties of H175G and of its complexes with imidazole. Combined with the crystal structures for these complexes, the data provide a detailed and consistent account of the modes of Im binding in the Hl 75G cavity and their dependence on buffer and pH. UV—vis, EPR, and resonance Raman spectra reveal multiple coordination states for H175G/Jm which can be correlated with the crystal structures to assign the following heme environments: H175G/H20/H20, H175G/Jmd/phosphate~, H175G/1nijH2O~, H175G/JmC/H,Od, and Hl75GIJm~IOH~, where H175G/X/Y defines the proximal species as X and the distal species as Y and c and d subscripts refer, where known, to the coordinated and dissociated states, respectively. Resonance Raman data for reduced H175G/Im show two substates for heme-coordinated Im differing in the strength of their hydrogen bond to Asp-235, in a fashion similar to WT CCP. NO binding to ferrous H175G/Im results in dissociation of Im from the heme but not from the cavity, while no dissociation is observed for WT CCP, indicating that steric tethering may, in part, control NO-induced dissociation of trans ligands. H175G/Im forms an oxidized compound I state with two distinct radical species, each with a dramatically different anisotropy and spin relaxation from that of the Trp-191 radical of WT CCP. It is suggested that these signals arise from alternate conformations of Trp 191 having different degrees of exchange coupling to the ferryl heme, possibly mediated by the conformational heterogeneity of Im within the H175G cavity. The kinetics of the reaction of H175G/Jm with H202 are multiphasic, also reflecting the multiple coordination states of Im. The rate of the fastest phase is essentially identical to that of WT CCP, indicating that the H175G/JmjH2Od state is fully reactive with peroxide. However, the overall rate of enzyme turnover using cytochrome c as a substrate is <5% of WT and is unaffected by Im coordination. In summary, Im coordination to H175G results in a number of conformers, one of which is structurally and spectroscopically very similar to WT CCP. However, while this form is fully reactive with peroxide, the reaction with cytochrome c remains inefficient, perhaps implicating the altered Trp- 191 radical species.
机译：咪唑不能补充酵母细胞色素C过氧化物酶的轴向组氨酸缺失突变体H175G中的功能，这是一个引人入胜但尚未解决的问题，它影响了我们对轴向配体在血红素催化中的作用的理解。在这里，我们报告H175G及其与咪唑配合物的功能和光谱性质。结合这些配合物的晶体结构，数据提供了对H75G空腔中Im结合模式及其对缓冲液和pH的依赖性的详细一致的说明。 UV-vis，EPR和共振拉曼光谱揭示了H175G / Jm的多个配位状态，这些状态可以与晶体结构关联以分配以下血红素环境：H175G / H20 / H20，H175G / Jmd /磷酸盐〜，H175G / 1nijH2O〜 ，H175G / JmC / H，Od和Hl75GIJm〜IOH〜，其中H175G / X / Y将近端物种定义为X，将远端物种定义为Y和c，而下标在已知的情况下是指配位和解离状态，分别。降低的H175G / Im的共振拉曼数据显示，血红素配位的Im的两个亚状态与Asp-235的氢键强度不同，与WT CCP相似。 NO与H175G / Im亚铁的结合会导致Im从血红素解离，但不会从腔中解离，而WT CCP则未观察到解离，这表明空间束缚可能部分控制了NO诱导的反式配体解离。 H175G / Im形成具有两个不同自由基基团的氧化的I状态，每个自由基基团的各向异性和自旋弛豫与WT CCP的Trp-191自由基大不相同。建议这些信号来自具有与亚铁血红素偶联的不同交换程度的Trp 191的交替构象，可能由H175G腔内Im的构象异质性介导。 H175G / Jm与H2O2反应的动力学是多相的，也反映了Im的多个配位态。最快相的速率与WT CCP的速率基本相同，这表明H175G / JmjH2Od状态可与过氧化物完全反应。但是，以细胞色素c为底物的酶总转化率小于WT的5％，不受Im配比的影响。总而言之，与H175G的Im配位导致许多构象异构体，其中之一在结构和光谱上与WT CCP非常相似。然而，尽管这种形式可与过氧化物完全反应，但与细胞色素c的反应仍然无效，这可能暗示了Trp-191自由基种类的改变。

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