Identification of bovine heart cytochrome c oxidase subunits modified by the lipid peroxidation product 4-hydroxy-2-nonenal
Identification of bovine heart cytochrome c oxidase subunits modified by the lipid peroxidation product 4-hydroxy-2-nonenal
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DOI：
10.1021/bi025896u
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发表时间：
2002-06-25
期刊：
影响因子：
2.9
通讯作者：
Robinson, NC
中科院分区：
文献类型：
--
作者：
Musatov, A;Carroll, CA;Robinson, NC
脂质过氧化产物4-羟基-2-壬烯醛（HNE）对牛心肌细胞色素c氧化酶（CcO）的失活具有时间和浓度依赖性，并符合拟一级动力学。细胞色素c氧化酶电子传递活性下降了50%，当酶在室温下孵育2小时，过量的HNE（300-500 μ M）。HNE修饰的CcO亚基通过两种质谱方法鉴定：电喷雾电离质谱（ESI/MS）和基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF/MS）。所有的实验测定的分子量与公布的序列值非常一致，小于20 kDa的亚基的精确度接近于每10000质量单位1份，大于20 kDa的三个亚基的精确度接近于每1000质量单位1份。两种MS方法均检测到6个CcO亚基与HNE反应后质量增加156 Da（亚基II、IV、Vb、VIIa、VIIc和VIII）;该结果表明每个亚基内赖氨酸或组氨酸残基上存在单个Michael型反应位点。HNE与亚基VIIc或亚基VIII（分别修饰为30%和50- 75%）的反应肯定是CcO抑制的原因。其他亚基的修饰都不超过5%，不能解释观察到的活性损失。HNE与VIII亚基的His-36的反应与CcO的50%抑制率最接近：（1）VIII亚基被HNE修饰的程度高于其他亚基;（2）VIII亚基修饰的时间依赖性与CcO的抑制率一致;（3）通过串联质谱分析，确定His-36为VIII亚基中HNE修饰的氨基酸残基。
Bovine heart cytochrome c oxidase (CcO) was inactivated by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) in a time- and concentration-dependent manner with pseudo-first-order kinetics. Cytochrome c oxidase electron transport activity decreased by as much as 50% when the enzyme was incubated for 2 h at room temperature with excess HNE (300-500 muM). HNE-modified CcO subunits were identified by two mass spectrometric methods: electrospray ionization mass spectrometry (ESI/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). All of the experimentally determined molecular masses were in excellent agreement with published sequence values with an accuracy of similar to1 part per 10000 mass units for subunits smaller than 20 kDa and similar to1 part per 1000 mass units for the three subunits larger than 20 kDa. Both MS methods detected six CcO subunits with an increased mass of 156 Da after reaction with HNE (subunits II, IV, Vb, VIIa, VIIc, and VIII); this result indicates a single Michael-type reaction site on either a lysine or histidine residue within each subunit. Reaction of HNE with either subunit VIIc or subunit VIII (modified similar to30% and 50-75%, respectively) must be responsible for CcO inhibition. None of the other subunits were modified more than 5% and could not account for the observed loss of activity. Reaction of HNE with His-36 of subunit VIII is most consistent with the similar to50% inhibition of CcO: (1) subunit VIII is modified more than any other subunit by HNE, (2) the time dependence of subunit VIII modification is consistent with the percent inhibition of CcO; (3) His-36 was identified as the HNE-modified amino acid residue within subunit VIII by tandem MS analysis.