Mechanistsic Studies on the Catalase Reactions: Introduction of Catalase Activity into Heme Proteins.

利用肌红蛋白突变体对过氧化氢酶反应进行了详细研究。Mb突变体的化合物I（Mb-I），<IV>与卟啉自由基阳离子配对的铁基物质（Fe^ =O），容易通过与接近化学计量量的间氯过苯甲酸（mCPBA）反应来制备。在Mb-1溶液中加入H_2O_2后，Mb-1被还原回铁态，不生成任何中间体。这表明Mb-Ⅰ具有双电子氧化H_2O_2的能力。用GC-MS分析了H_218O_2/H_2^O_2（50：50）混合液<16>中H_64D或F43 H/H64 L Mb放出的O_2，结果表明生成了~（18）O_2（m/e=36）和~（16）O_2（m/e = 32），但没有生成^<16>O^<18>O（m/e = 34）。这表明O_2是由H_2O_2在不破坏O-O键的情况下通过双电子氧化形成的。氘同位素对Mb突变体和过氧化氢酶的过氧化氢反应的影响表明，溶壁微球菌过氧化氢酶（MLC）和F43 H/H64 L Mb的过氧化氢反应是通过离子机制进行的，同位素效应小于4.0，因为远端组氨酸残基位于适当的位置，作为离子反应的一般酸碱催化剂。与此相反，其他Mb突变体如H64 X（X：A，S，D）和L29 H/H64 L Mb通过自由基机制氧化H_2O_2，在10 - 29的范围内，由于缺乏一般的酸碱催化剂，Mb-I具有很大的同位素效应

The catalase reaction has been studied in detail by using myoglobin (Mb) mutants. Compound I of Mb mutants (Mb-I), a ferryl species (Fe^<IV>=O) paired with a porphyrin radical cation, is readily prepared by the reaction with a nearly stoichiometric amount of m-chloroperbenzoic acid (mCPBA). Upon the addition of H_2O_2 to an Mb-I solution, Mb-1 is reduced back to the ferric state without forming any intermediates. This indicates that Mb-I is capable of performing two-electron oxidation of H_2O_2 (catalatic reaction). GC-MS analysis of the evolved O2 from a 50:50 mixture of H_218O_2/H_2^<16>O_2 solution containing H64D or F43H/H64L Mb showed the formation of 18O2 (m/e=36) and 16O2 (m/e = 32) but not ^<16>O^<18>O (m/e = 34). This implies that O2 is formed by two electron oxidation of H_2O_2 without breaking the O-O bond. Deuterium isotope effects on the catalatic reactions of Mb mutants and catalase suggest that the catalatic reactions of Micrococcus lysodeikticus Catalase (MLC) and F43H/H64L Mb proceed via an ionic mechanism with a small isotope effect of less than 4.0, since the distal histidine residue is located at a proper position to act as a general acid-base catalyst for the ionic reaction. In contrast, other Mb mutants such as H64X (X: A, S, and D) and L29H/H64L Mb oxidize H_2O_2 via a radical mechanism in which a hydrogen atom is abstracted by Mb-I with a large isotope effect in a range of 10 to 29, due to a lack of the general acid-base catalyst