Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, Kolkata, India.
Arch Biochem Biophys. 2010 Mar 15;495(2):129-35. doi: 10.1016/j.abb.2010.01.002. Epub 2010 Jan 12.
Architecture of hemoprotein is solely responsible for different nature of heme coordination. Here we report that substitution of the acidic surface residue Glu226 to Ala in ascorbate peroxidase from Leishmania major alters the 5 coordinate high spin (5cHS) to a 6 coordinate low spin (6cLS) form at pH 7.5. Using UV-visible spectrophotometry, we show that the sixth ligand of heme in Glu226Ala at pH 7.5 is hydroxo. When the pH is decreased to 5.5, a new species of Glu226Ala appeared that had a spectrum characteristic of a 6cHS derivative. Stopped flow spectrophotometric techniques revealed that characteristics of Compound I was not seen in the Glu226Ala in presence of H(2)O(2). Similarly guaiacol, ascorbate and ferrocytochrome c oxidation rate was 10(3) orders less for the Glu226Ala mutants compared to the wild type. These data suggested that surface acidic residue Glu226 might play role in proper maintenance of active site conformation.
血红素蛋白的结构完全负责血红素配位的不同性质。在这里，我们报告说，在 pH 值为 7.5 时，将主要利什曼原虫抗坏血酸过氧化物酶中的酸性表面残基 Glu226 替换为 Ala，会将 5 配位高自旋（5cHS）转变为 6 配位低自旋（6cLS）形式。我们使用紫外可见分光光度法表明，在 pH 值为 7.5 时，Ala 中的 Glu226 的血红素的第六个配体是羟。当 pH 值降低到 5.5 时，出现了一种新的 Glu226Ala 物种，其光谱特征为 6cHS 衍生物。停流分光光度技术表明，在 H(2)O(2)存在下，Glu226Ala 中未观察到化合物 I 的特征。类似地，与野生型相比，Glu226Ala 突变体的愈创木酚、抗坏血酸和细胞色素 c 氧化速率低 10(3) 个数量级。这些数据表明，表面酸性残基 Glu226 可能在适当维持活性部位构象方面发挥作用。
