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

利用肌红蛋白（Mb）突变体对过氧化氢酶反应进行了详细的研究。Mb突变体的化合物I （Mb-I）是一种铁基（Fe^<IV>=O）与卟啉自由基阳离子配对，可以很容易地与接近化学计量量的间氯过苯甲酸（mCPBA）反应。在Mb-1溶液中加入H_2O_2后，Mb-1被还原回铁态，没有形成任何中间产物。这表明Mb-I能够对H_2O_2进行双电子氧化（催化反应）。用气相色谱-质谱分析H_218O_2/H_2^<16>O_2溶液中含有H64D或F43H/H64L Mb的50:50混合物中析出的O2，发现形成了18O2 （m/e=36）和16O2 (m/e= 32)，但没有形成^<16>O^<18>O （m/e= 34）。这表明O2是由H_2O_2的两个电子氧化而不破坏O-O键形成的。氘同位素对Mb突变体和过氧化氢酶催化反应的影响表明，溶血性微球菌过氧化氢酶（MLC）和F43H/H64L Mb的催化反应是通过离子机制进行的，同位素效应较小，小于4.0，因为远端组氨酸残基位于合适的位置，充当离子反应的一般酸碱催化剂。相比之下，其他Mb突变体如H64X （X: A， S和D）和L29H/H64L Mb由于缺乏一般的酸碱催化剂，通过自由基机制将H_2O_2氧化，其中氢原子被Mb- i抽离，其同位素效应在10 ~ 29范围内

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