Loewen P C, Switala J, von Ossowski I, Hillar A, Christie A, Tattrie B, Nicholls P
Department of Microbiology, University of Manitoba, Winnipeg, Canada.
Biochemistry. 1993 Sep 28;32(38):10159-64. doi: 10.1021/bi00089a035.
Catalase HPII from aerobically grown Escherichia coli normally contains heme d but cultures grown with poor or no aeration produce HPII containing a mixture of heme d and protoheme IX. The protoheme component of HPII from anaerobically grown cells is converted into heme d during treatment of the purified enzyme with hydrogen peroxide. It is concluded that heme d found in catalase HPII is formed by the cis-hydroxylation of protoheme in a reaction catalyzed by catalase HPII using hydrogen peroxide as a substrate. The distal His128 residue of HPII is absolutely required for the protoheme to heme d conversion. Two mutant enzymes, Ala128 and Asn128, are catalytically inactive and contain only protoheme, which is unaffected by hydrogen peroxide treatment. The Asn201 residue is not an absolute requirement for heme conversion. The mutant enzyme Ala201 contains predominantly heme d and is partially active. However, insertion of a histidyl residue to give the His201 enzyme interferes with the heme conversion reaction. This mutant form is isolated as a protoheme enzyme with limited activity, and a reversible conversion to a heme d-like species occurs in vitro in the presence of continuously generated hydrogen peroxide.
来自需氧生长的大肠杆菌的过氧化氢酶HPII通常含有血红素d，但在通气不良或无通气条件下生长的培养物会产生含有血红素d和原血红素IX混合物的HPII。厌氧生长细胞的HPII中的原血红素成分在用过氧化氢处理纯化酶的过程中会转化为血红素d。得出的结论是，过氧化氢酶HPII中发现的血红素d是由原血红素在过氧化氢酶HPII以过氧化氢为底物催化的反应中进行顺式羟基化形成的。HPII的远端His128残基是原血红素向血红素d转化绝对必需的。两种突变酶Ala128和Asn128无催化活性，仅含有原血红素，且不受过氧化氢处理的影响。Asn201残基不是血红素转化的绝对必需条件。突变酶Ala201主要含有血红素d且具有部分活性。然而插入一个组氨酸残基得到His201酶会干扰血红素转化反应。这种突变形式被分离为具有有限活性的原血红素酶，并且在持续产生过氧化氢的情况下，体外会发生向类似血红素d物种的可逆转化。
