Geobacter sulfurreducens cytochrome c peroxidases: electrochemical classification of catalytic mechanisms.
Geobacter sulfurreducens cytochrome c peroxidases: electrochemical classification of catalytic mechanisms.
复制标题
硫还原地杆菌细胞色素 c 过氧化物酶：催化机制的电化学分类。
DOI：
10.1021/bi200399h
复制
发表时间：
2011
期刊：
影响因子：
2.9
通讯作者：
Elliott,SeanJ
中科院分区：
文献类型：
--
作者：
Ellis,KatieE;Seidel,Julian;Einsle,Oliver;Elliott,SeanJ
细菌细胞色素过氧化物酶（CcP）是二血红素氧化还原蛋白，其将过氧化氢还原成水。它们的典型特征是一个过氧化物（称为L，“低还原电位”）活性位点血红素和一个与电子转移相关的次级血红素（H，“高还原电位”），以及一种仅在H-血红素被预还原到Fe Ⅱ氧化态时才存在的酶活性。预还原步骤导致活性位点本身的构象变化，其中含组氨酸的环将采用“开放”构象，允许过氧化氢与L-血红素的FeIII结合。值得注意的是，来自欧洲亚硝化单胞菌的酶不需要预还原。以前，我们已经表明，蛋白膜伏安法（PFV）是一个非常有用的工具，区分的电催化机制的硝基单胞菌型酶从其他CcPs。在这里，我们将PFV应用于最近描述的来自Geobactersulfurreducens和GeobacterS 134 P/V135 K双突变体的酶，它们已被证明分别类似于过氧化物酶的典型亚类和亚硝化单胞菌酶的亚类的成员。在这里，我们发现野生型GeobacterCcP确实与需要还原活化的细菌CcP在电化学上相似，但S134 P/V135 K突变体显示出两个电催化阶段：一种是低潜力的，像野生型酶，而第二种，高电位相，其电位依赖于底物结合和pH，但电位与H-血红素非常相似。这些发现被解释在一个模型，其中限速蛋白内电子转移支配的S134 P/V135 K酶的催化性能。
Bacterial cytochromecperoxidase (CcP) enzymes are diheme redox proteins that reduce hydrogen peroxide to water. They are canonically characterized by a peroxidatic (calledL, for “low reduction potential”) active site heme and a secondary heme (H, for “high reduction potential”) associated with electron transfer, and an enzymatic activity that exists only when theH-heme is prereduced to the FeIIoxidation state. The prereduction step results in a conformational change at the active site itself, where a histidine-bearing loop will adopt an “open” conformation allowing hydrogen peroxide to bind to the FeIIIof theL-heme. Notably, the enzyme fromNitrosomonas europaeadoes not require prereduction. Previously, we have shown that protein film voltammetry (PFV) is a highly useful tool for distinguishing the electrocatalytic mechanisms of theNitromonastype of enzyme from other CcPs. Here, we apply PFV to the recently described enzyme fromGeobacter sulfurreducensand theGeobacterS134P/V135K double mutant, which have been shown to be similar to members of the canonical subclass of peroxidases and theNitrosomonassubclass of enzymes, respectively. Here we find that the wild-typeGeobacterCcP is indeed similar electrochemically to the bacterial CcPs that require reductive activation, yet the S134P/V135K mutant shows two phases of electrocatalysis: one that is low in potential, like that of the wild-type enzyme, and a second, higher-potential phase that has a potential dependent upon substrate binding and pH yet is at a potential that is very similar to that of theH-heme. These findings are interpreted in terms of a model in which rate-limiting intraprotein electron transfer governs the catalytic performance of the S134P/V135K enzyme.