Nature of the inhibition of horseradish peroxidase and mitochondrial cytochrome c oxidase by cyanyl radical
Nature of the inhibition of horseradish peroxidase and mitochondrial cytochrome c oxidase by cyanyl radical
复制标题
DOI：
10.1021/bi992652
复制
发表时间：
2000-04-18
期刊：
影响因子：
2.9
通讯作者：
Mason, RP
中科院分区：
文献类型：
--
作者：
Chen, YR;Deterding, LJ;Mason, RP
先前的研究证实，氰基自由基(。CN)，检测为5,5-二甲基-1-吡咯啉n -氧化物(DMPO)/。利用电子自旋共振（ESR）自旋捕获技术，辣根过氧化物酶（HRP）在过氧化氢（H2O2）存在下和线粒体细胞色素c氧化酶（CcO）在H2O2不存在的情况下可以生成CN。为了研究氰基自由基对CN-的抑制机制，我们分别从HRP和CcO反应中分离出原卟啉铁IX和血红素a，并对其进行了表征。在基质辅助激光解吸/电离（MALDI）质谱中，通过观察M /z = 642.9的质子化分子（M + H）（+），确定了从HRP/H2O2/KCN反应混合物中纯化的血红素为氰血红素。联吡啶亚铁氰血红素配合物的质子核磁共振谱显示，四个介孔质子中有一个缺失，并被一个氰基取代，表明单一的血红素衍生产物是中位氰血红素。由中位氰血红素与HRP脱酶（apoHRP）重组而来的HRP全酶未显示出可检测到的催化活性。蓝血红素重组的apoHRP的Soret峰由天然HRP的403 nm峰移至411 nm。相比之下，从部分或完全抑制的CcO中分离的血红素a，其MALDI质谱显示（M + H）（+）为M /z = 853.6，并通过其吡啶色素谱显示，原卟啉IX的结构没有任何变化。然而，在CcO与氰化物的反应中，可以检测到CcO上一个以蛋白质为中心的自由基，根据n-乙基马来酰亚胺预处理对其形成的抑制，鉴定为巯基自由基，这表明受到氰基自由基攻击的不是原卟啉IX，而是蛋白质基质。除了HRP和CcO的血红素结构不同外，现有的晶体学数据还表明，不同的血红素环境可能导致HRP和CcO受到氰基自由基的不同抑制机制。
Previous studies established that the cyanyl radical (.CN), detected as 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/.CN by the electron spin resonance (ESR) spin-trapping technique, can be generated by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H2O2) and by mitochondrial cytochrome c oxidase (CcO) in the absence of H2O2 TO investigate the mechanism of inhibition by cyanyl radical, we isolated and characterized the iron protoporphyrin IX and heme a from the reactions of CN- with HRP and CcO, respectively. The purified heme from the reaction mixture of HRP/H2O2/KCN was unambiguously identified as cyanoheme by the observation of the protonated molecule, (M + H)(+), of m/z = 642.9 in the matrix-assisted laser desorption/ionization (MALDI) mass spectrum. The proton NMR spectrum of the bipyridyl ferrous cyanoheme complex revealed that one of the four meso protons was missing and had been replaced with a cyanyl group, indicating that the single, heme-derived product was meso-cyanoheme. The holoenzyme of HRP from the reconstitution of meso-cyanoheme with the apoenzyme of HRP (apoHRP) showed no detectable catalytic activity. The Soret peak of cyanoheme-reconstituted apoHRP was shifted to 411 nm from the 403 nm peak of native HRP. In contrast, the heme a isolated from partially or fully inhibited CcO did not show any change in the structure of the protoporphyrin IX as indicated by its MALDI mass spectrum, which showed an (M + H)(+) of m/z = 853.6, and by its pyridine hemochromogen spectrum. However, a protein-centered radical on the CcO can be detected in the reaction of CcO with cyanide and was identified as the thiyl radical(s) based on inhibition of its formation by N-ethylmaleimide pretreatment, suggesting that the protein matrix rather than protoporphyrin IX was attacked by the cyanyl radical. In addition to the difference in heme structures between HRP and CcO, the available crystallographic data also suggested that the distinct heme environments may contribute to the different inhibition mechanisms of HRP and CcO by cyanyl radical.