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)是一种铁基物种(Fe^&lt；IV&gt；=O)，与卟啉自由基阳离子配对，通过与接近化学计量比的间氯过苯甲酸(MCPBA)反应很容易得到。在Mb-I溶液中加入H_2O_2后，Mb-1被还原回铁态，而不形成任何中间体。这表明Mb-I能够进行H_2O_2的双电子氧化(过氧化氢反应)。GC-MS分析表明，含有H64D或F43H/H64L Mb的H_218O_2/H_2^&lt；16&gt；O_2混合溶液50：50放出的O2有18O_2(m/e=36)和16O_2(m/e=32)，但没有^&lt；16&gt；O^&lt；18&gt；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。氢同位素对Mb突变体和过氧化氢酶催化反应的影响表明，溶血微球菌过氧化氢酶(MLC)和F43H/H64L Mb的催化反应是通过离子机理进行的，其小同位素效应小于4.0，因为远端组氨酸残基位于合适的位置，可以作为离子反应的普通酸碱催化剂。相反，其他Mb突变体，如H64X(X：A，S和D)和L29H/H64L Mb，由于缺乏通用的酸碱催化剂，通过自由基机制氧化H_2O_2，其中氢原子被具有10到29范围内的大同位素效应的Mb-I提取

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