CHARACTERIZATION OF UNSTABLE INTERMEDIATES OF HEME ENZYMES BY UTILIZING ENZYMES AND THEIR HYBRIDES

聚乙二醇化辣根过氧化物酶(PEGHRP)在有机溶剂和水溶液中均能催化单电子和双电子氧化反应。尽管愈创木酚在苯和氯苯中的氧化速度比在磷酸盐缓冲溶液中慢5个数量级，但化合物I和II也参与了有机介质中的催化循环。因子分析和快速扫描数据集的全局拟合表明，化合物I在有机介质中的形成包括两个步骤：第一个快过程和第二个慢过程，这表明催化循环中有一个H_2O_2-HRP络合物参与。化合物I的不稳定前体在-20ﾟC下与过氧化氢在氯苯中反应后稳定下来，其吸收光谱不具有高卟啉光谱的特征，但具有R38L-HRP中观察到的正常吸收光谱。更重要的是，化合物I在氯苯中能在-20ﾟC下保持1小时以上。我们发现，相对于过氧化物酶和野生型Mb，将Mb的远端组氨酸从位置移动到43位会增加过氧化物酶的活性。更重要的是，一种类似铁基的阳离子物种首次被鉴定为Mb突变体的催化中间体。讨论了远端组氨酸的排列与血色素蛋白功能的相关性。

Polyethylene glycolated horseradish peroxidase (PEG-HRP) can catalyze one- and two-electron oxidation reactions in organic solvents as well as in aqueous buffer. Even though the oxidation of guaiacol in benzene and chlorobenzene is five order of magnitude slower than in phosphate buffer, compound I and II are also involved in the catalytic cycle in organic media. Factor analysis and global fittings of rapid scan data set reveal that the formation of compound I of PEG-HRP in organic media consists of two steps : the first fast and the second slow process, and suggest the involvement of a H2O2-HRP complex in the catalytic cycle. The labile precursor of compound I is stabilized when PEG-HRP reacts with hydrogen peroxide in chlorobenzene at -20ﾟC.The absorption spectrum of the precursor does not exhibit the features of hyperporphyrin spectrum but has a normal Soret as previously observed in R38L HRP.More importantly, compound I of PEG-HRP can be maintained for more than an hour at -20ﾟC in chlorobenzene.Comparison of the X-ray crystal structures of sperm whale myoglobin (Mb) and cytochrome c peroxidase (CcP) allows us to design Mb mutants to mimic the active site of peroxidase. We have found that relocating the distal histidine of Mb from position 64 to 43 increases peroxygenase activities with respect to those of peroxidase as well as wild type Mb. More importantly, a ferryl radical cation like species has been identified as the catalytic intermediate of the Mb mutant for the first time. The relevance of the alignment of distal histidine to the functions of hemoproteins is discussed.