Medical Research Council-Mitochondrial Biology Unit, University of Cambridge, Cambridge CB2 0XY, UK.
Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK.
Cell Chem Biol. 2022 Apr 21;29(4):636-649.e14. doi: 10.1016/j.chembiol.2021.10.010. Epub 2021 Nov 4.
Mammalian complex I can adopt catalytically active (A-) or deactive (D-) states. A defining feature of the reversible transition between these two defined states is thought to be exposure of the ND3 subunit Cys39 residue in the D-state and its occlusion in the A-state. As the catalytic A/D transition is important in health and disease, we set out to quantify it by measuring Cys39 exposure using isotopic labeling and mass spectrometry, in parallel with complex I NADH/CoQ oxidoreductase activity. To our surprise, we found significant Cys39 exposure during NADH/CoQ oxidoreductase activity. Furthermore, this activity was unaffected if Cys39 alkylation occurred during complex I-linked respiration. In contrast, alkylation of catalytically inactive complex I irreversibly blocked the reactivation of NADH/CoQ oxidoreductase activity by NADH. Thus, Cys39 of ND3 is exposed in complex I during mitochondrial respiration, with significant implications for our understanding of the A/D transition and the mechanism of complex I.
哺乳动物复合物 I 可以采用催化活性 (A-) 或非活性 (D-) 状态。人们认为，这两种确定状态之间可逆转换的一个定义特征是 D 状态下 ND3 亚基 Cys39 残基的暴露及其在 A 状态下的封闭。由于催化 A/D 转换在健康和疾病中很重要，我们通过使用同位素标记和质谱法测量 Cys39 的暴露来定量它，同时测量复合物 I NADH/CoQ 氧化还原酶活性。令我们惊讶的是，我们发现 NADH/CoQ 氧化还原酶活性过程中有明显的 Cys39 暴露。此外，如果在复合物 I 连接的呼吸过程中发生 Cys39 烷基化，这种活性不会受到影响。相比之下，催化非活性复合物 I 的烷基化会不可逆地阻止 NADH 对 NADH/CoQ 氧化还原酶活性的再激活。因此，ND3 的 Cys39 在线粒体呼吸过程中暴露于复合物 I 中，这对我们理解 A/D 转换和复合物 I 的机制具有重要意义。
