An electron spin resonance spin-trapping investigation of the free radicals formed by the reaction of mitochondrial cytochrome c oxidase with H2O2
An electron spin resonance spin-trapping investigation of the free radicals formed by the reaction of mitochondrial cytochrome c oxidase with H2O2
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DOI：
10.1074/jbc.274.6.3308
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发表时间：
1999-02-05
影响因子：
4.8
通讯作者：
Mason, RP
中科院分区：
文献类型：
--
作者：
Chen, YR;Gunther, MR;Mason, RP
采用ESR自旋俘获技术研究了纯化牛线粒体细胞色素c氧化酶（CcO）与过氧化氢的反应。以蛋白质为中心的自由基加合物被5,5-二甲基-1-吡啶N-氧化物捕获，并根据其超精细偶联常数A (N) = 14.7 G和A (β)(H) = 15.7 G被确定为巯基自由基加合物。利用亚硝基自旋陷阱3,5-二溴-4-亚硝基苯磺酸（DBNBS）和2-甲基-2-亚硝基丙烷（MNP）获得的ESR光谱表明，DBNBS/CcO和MNP/。CcO自由基加合物是由捕获蛋白质来源的自由基形成的固定化氮氧化物。非特异性蛋白酶处理MNP-d(9)/(NEM)- n -时，游离巯基与n -乙基马来酰亚胺（NEM）的烷基化作用阻止了5,5 -二甲基-1-吡咯啉n -氧化物加合物的形成，改变了MNP和DBNBS自由基加合物的光谱。-CcO将其固定态氮氧化物的光谱转换为具有快速分子运动特征的各向同性三线光谱，在该光谱中检测到超精细耦合，并将其分配给MNP/。用氰化钾抑制CcO或NEM-CcO均可阻止MNP加合物的形成，表明血红素参与了反应。结果表明，一个或多个半胱氨酸残基是假定的铁酰基卟啉阳离子残基中间体的首选还原剂。当半胱氨酸残基被NEM阻断时，一个或多个酪氨酸残基成为首选还原剂，形成酪氨酸自由基。
The reaction of purified bovine mitochondrial cytochrome c oxidase (CcO) and hydrogen peroxide was studied using the ESR spin-trapping technique. A protein-centered radical adduct was trapped by 5,5-dimethyl-1-pyrroline N-oxide and was assigned to a thiyl radical adduct based on its hyperfine coupling constants of a(N) = 14.7 G and a(beta)(H) = 15.7 G. The ESR spectra obtained using the nitroso spin traps 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and 2-methyl-2-nitrosopropane (MNP) indicated that both DBNBS/.CcO and MNP/.CcO radical adducts are immobilized nitroxides formed by the trapping of protein-derived radicals. Alkylation of the free thiols on the enzyme with N-ethylmaleimide (NEM) prevented 5,5 dimethyl-1-pyrroline N-oxide adduct formation and changed the spectra of the MNP and DBNBS radical adducts, Nonspecific protease treatment of MNP-d(9)/(NEM)-N-.-CcO converted its spectrum from that of an immobilized nitroxide to an isotropic three-line spectrum characteristic of rapid molecular motion, Super-hyperfine couplings were detected in this spectrum and assigned to the MNP/.tyrosyl adduct(s), The inhibition of either CcO or NEM-CcO with potassium cyanide prevented detectable MNP adduct formation, indicating heme involvement in the reaction. The results indicate that one or more cysteine residues are the preferred reductant of the presumed ferryl porphyrin cation radical residue intermediate. When the cysteine residues are blocked with NEM, one or more tyrosine residues become the preferred reductant, forming the tyrosyl radical.