Mutagenesis of a proton linkage pathway in Escherichia coli manganese superoxide dismutase.
Mutagenesis of a proton linkage pathway in Escherichia coli manganese superoxide dismutase.
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大肠杆菌锰超氧化物歧化酶中质子连接途径的诱变。
DOI：
10.1021/bi9704212
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Whittaker,JW
中科院分区：
文献类型：
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作者：
Whittaker,MM;Whittaker,JW
大肠杆菌锰超氧化物歧化酶（MnSD）的突变表明，严格保守的网关酪氨酸（Y34）参与外源配体相互作用。苯丙氨酸（Y34 F）的保守取代这一残基的影响pH敏感性的活性位点的金属离子和扰动配体结合，稳定的温度无关的六配位叠氮化物复合物。突变复合物的特征在于光学和电子顺磁共振（EPR）光谱是不同的，从相应的野生型形式和阴离子亲和力的改变，与修改的金属配体的碱性一致。然而，歧化酶活性仅因诱变而略微降低，这意味着酪氨酸-34对于催化不是必需的，并且可能间接地作为用于周转的质子供体起作用，与金属配体的质子化循环偶联。对于wt酶，将活性位点的pH依赖性转变从9.7（Mn）移动到6.4（Fe）。这种pH-偶联的转变再次转变为Y34 F Fe 2-MnSD的更高有效pKa，允许突变体在生理pH范围内具有催化活性，并降低酶的金属选择性。过氧化物的Fe络合物的敏感性是不同的野生型和突变体蛋白质，表明Y34在过氧化物相互作用的作用。这些结果为超氧化物歧化酶中保守的过氧化物-质子化连接途径提供了证据，类似于过氧化物酶的质子中继链，并表明Mn和Fe超氧化物歧化酶的选择性是由与金属配体的质子偶联决定的。
Mutagenesis ofEscherichia colimanganese superoxide dismutase (MnSD) demonstrates involvement of the strictly conserved gateway tyrosine (Y34) in exogenous ligand interactions. Conservative replacement of this residue by phenylalanine (Y34F) affects the pH sensitivity of the active-site metal ion and perturbs ligand binding, stabilizing a temperature-independent six-coordinate azide complex. Mutant complexes characterized by optical and electron paramagnetic resonance (EPR) spectroscopy are distinct from the corresponding wild-type forms and the anion affinities are altered, consistent with modified basicity of the metal ligands. However, dismutase activity is only slightly reduced by mutagenesis, implying that tyrosine-34 is not essential for catalysis and may function indirectly as a proton donor for turnover, coupled to a protonation cycle of the metal ligands.In vivosubstitution of Fe for Mn in the MnSD wild-type and mutant proteins leads to increased affinity for azide and altered active-site properties, shifting the pH-dependent transition of the active site from 9.7 (Mn) to 6.4 (Fe) for wt enzyme. This pH-coupled transition shifts once more to a higher effective pKafor Y34F Fe2-MnSD, allowing the mutant to be catalytically active well into the physiological pH range and decreasing the metal selectivity of the enzyme. Peroxide sensitivities of the Fe complexes are distinct for the wild-type and mutant proteins, indicating a role for Y34 in peroxide interactions. These results provide evidence for a conserved peroxide−protonation linkage pathway in superoxide dismutases, analogous to the proton relay chains of peroxidases, and suggests that the selectivity of Mn and Fe superoxide dismutases is determined by proton coupling with metal ligands.