Pearce Linda L, Kanai Anthony J, Epperly Michael W, Peterson Jim
Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA.
Nitric Oxide. 2005 Dec;13(4):254-63. doi: 10.1016/j.niox.2005.07.010. Epub 2005 Sep 23.
The effects of both nitric oxide (NO) and peroxynitrite on complexes I (NADH dehydrogenase) and III (cytochrome c reductase) isolated from bovine heart have been examined. EPR signals ("g=2.01") previously detected in association with loss of complex I and III activities in cultured cells and isolated mitochondria subjected to nitrosative stress are shown not to arise from these particular enzymes. Neither NO nor peroxynitrite (ONO(2)(-)) reacts to any appreciable extent with the oxidized forms of flavin mononucleotide, iron-sulfur clusters, or heme moieties found in complexes I and III. However, ONO(2)(-) is readily able to abstract electrons from the reduced forms of both complexes I and III, without any apparent modification of the enzyme cofactors. While no attempt was made in the present study to catalog all the possible modifications, it is clear that ONO(2)(-) can react with the protein moieties of the enzymes. For example, when added in excess, ONO(2)(-) derivatizes a select few tyrosine residues in both complexes I and III forming 3-nitrotyrosine as detected by immunoblots. In the case of complex I, we find a minimum of 3 out of the 46 subunits present were modified (49, approximately 18, and approximately 15kDa); whereas in complex III, 4 out of the 13 subunits stained for 3-nitrotyrosine (46, 27, 7, and 6kDa). Significant irreversible inhibition of activity required the addition of >10(2)-fold excesses of ONO(2)(-) to the enzymes. At 10(3)-fold excess of added ONO(2)(-), the activity of complex I was only diminished by approximately 18%, while a 60% loss of activity was observed for complex III.
已经研究了一氧化氮（NO）和过氧亚硝酸盐对从牛心脏分离出的复合物I（NADH脱氢酶）和复合物III（细胞色素c还原酶）的影响。先前在受到亚硝化应激的培养细胞和分离的线粒体中，与复合物I和III活性丧失相关联检测到的电子顺磁共振信号（“g = 2.01”）表明并非源自这些特定的酶。NO和过氧亚硝酸盐（ONO₂⁻）均未与复合物I和III中发现的黄素单核苷酸、铁硫簇或血红素部分的氧化形式发生任何明显反应。然而，ONO₂⁻能够轻易地从复合物I和III的还原形式中夺取电子，而酶辅因子没有任何明显的修饰。虽然本研究未尝试对所有可能的修饰进行分类，但很明显ONO₂⁻可以与酶的蛋白质部分发生反应。例如，当过量添加时，ONO₂⁻会使复合物I和III中的少数酪氨酸残基衍生化，通过免疫印迹检测形成3-硝基酪氨酸。就复合物I而言，我们发现46个亚基中至少有3个被修饰（49kDa、约18kDa和约15kDa）；而在复合物III中，13个亚基中有4个被染成3-硝基酪氨酸（46kDa、27kDa、7kDa和6kDa）。活性的显著不可逆抑制需要向酶中添加超过10²倍过量的ONO₂⁻。当添加的ONO₂⁻过量10³倍时，复合物I的活性仅降低约18%，而复合物III的活性则降低了60%。
