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Molecular Mechanisms of Heme Degradation

血红素降解的分子机制

基本信息

批准号：
62480125
负责人：
ORII Yutaka
金额：
$ 3.78万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

The reaction mechanism of conversion of oxyprotoheme IX to verdohemochrome IX in heme degradation was investigated by employing purified preparations of heme oxygenase and NADPH-cytochrome c reductase in homogeneous state and synthetic -oxyprotoheme IX and its isomers. The reaction was followed kinetically as difference spectral changes and the reaction products were analyzed by HPLC.Spectral examinations and product analyses clearly demonstrated that only -isomer of oxyprotoheme IX was degraded efficiently to biliverdin IX. In the reconstituted reconstituted reaction system K_m for -oxyprotoheme IX was 3.6 Malthough this was almost two-fold that for the natural substrate, protoheme IX. The rate of conversion of -oxyprotoheme IX to biliverdin IX was two times faster than that of protoheme IX. These kinetic parameters strongly support a previous proposal that -oxyprotoheme IX is an intermediate during heme degradation.The activity of bovine heart cytochrome oxidase decreased when the enzyme was illuminated in the presence of protoporphyrin IX under aerobic conditions. The photoinactivation of the enzyme was dependent on the protoporophyrin concentration andillumination time, but the photodynamic effect was not observed under atmosphere of 100% nitrogen. The generation of singlet oxygen was confirmed by ESR spectroscopy as the appearance of nitroxide radical from 2,2,6,6-tetramethyul-4-peperidone. Scavengers of singlet oxygen like histidine, GMP or DABCO were effective in preventing the enzyme from photoinactivation. Thus singlet oxygen was concluded to be deleterious to cytochrome oxidase and degrade heme a moiety as revealed by spectral examinations.

采用均相纯化制备血红素加氧酶和nadph -细胞色素c还原酶，合成-氧原血红素IX及其同分异构体，研究血红素降解过程中氧原血红素IX转化为紫色素IX的反应机理。对反应进行了动力学跟踪，得到了不同光谱的变化，并对反应产物进行了高效液相色谱分析。光谱检查和产物分析清楚地表明，氧原血红素IX只有-异构体被有效地降解为胆绿素IX。在重建的重建反应体系中-氧原血红素IX的K_m为3.6 m，尽管这几乎是天然底物原血红素IX的两倍。-氧原血红素IX转化为胆绿素IX的速度比原血红素IX快2倍。这些动力学参数有力地支持了先前提出的-氧原血红素IX是血红素降解过程中的中间体。在有氧条件下，原卟啉IX照射牛心脏细胞色素氧化酶的活性降低。该酶的光失活与原卟啉浓度和光照时间有关，但在100%氮气氛下未观察到光动力效应。单线态氧的生成是由2,2,6,6-四甲基-4-培立酮的氮氧化物自由基的出现证实的。单线态氧清除剂如组氨酸、GMP或DABCO能有效阻止酶的光失活。因此，单线态氧对细胞色素氧化酶和血红素有一定的降解作用。