High rates of superoxide production in skeletal-muscle mitochondria respiring on both complex I- and complex II-linked substrates
High rates of superoxide production in skeletal-muscle mitochondria respiring on both complex I- and complex II-linked substrates
复制标题
DOI：
10.1042/bj20071162
复制
发表时间：
2008-01-15
影响因子：
4.1
通讯作者：
Van Remmen, Holly
中科院分区：
文献类型：
--
作者：
Muller, Florian L.;Liu, Yuhong;Van Remmen, Holly
尽管对超氧化物作为病理学的潜在原因有相当大的兴趣，但其由线粒体产生的有害物质的机制仍然知之甚少。先前在纯化线粒体中的研究已经发现，在通过复合物I的琥珀酸盐驱动的反向电子转移中观察到超氧化物产生的最高速率，尽管该途径的生理重要性存在争议，因为它需要高浓度的琥珀酸盐，并且被认为当NAD处于还原状态时不会发生。然而，很少有研究已经检查过的超氧化物的生产率与线粒体呼吸的NADH连接（如谷氨酸）和复杂的II连接的基板。在本研究中，我们发现谷氨酸盐+琥珀酸盐（类似于1100 pmol H2 O2中心点min（-1）mg（-1））的超氧化物产生速率（以H2 O2间接测量）出乎意料地远高于琥珀酸盐（类似于400 pmol H2 O2中心点min（-1））。mg（-1））或谷氨酸盐（类似于80 pmol H2 O2中心点min（-1）。mg（-1））。谷氨酸盐+琥珀酸盐的超氧化物产量即使在低底物浓度（< 1 mM）下也保持较高水平，鱼藤酮可降低超氧化物产量，FCCP（羰基氰化物对三氟甲氧基苯腙）可完全消除超氧化物产量，这表明超氧化物产量在很大程度上必须来源于通过复合物I的反向电子转移。当谷氨酸被丙酮酸、α-酮戊二酸或棕榈酰肉毒碱取代时，得到了类似的结果。相比之下，通过添加苹果酸盐（苹果酸盐+琥珀酸盐-30 pmol的H2 O2- min（-1）- mg（-1）），超氧化物产生持续降低。我们建议，苹果酸对超氧化物的产生的抑制作用可以解释草酰乙酸抑制复合物II。总之，本研究结果表明，逆电子转移介导的超氧化物的生产可以发生在生理上现实的底物条件下，并建议草酰乙酸抑制复合物11可能是一种自适应机制，以尽量减少这一点。
Despite the considerable interest in superoxide as a potential cause of pathology, the mechanisms of its deleterious production by mitochondria remain poorly understood. Previous studies in purified mitochondria have found that the highest rates of superoxide production are observed with succinate-driven reverse-electron transfer through complex I, although the physiological importance of this pathway is disputed because it necessitates high concentrations of succinate and is thought not to occur when NAD is in the reduced state. However, very few studies have examined the rates of superoxide production with mitochondria respiring on both NADH-linked (e.g. glutamate) and complex II-linked substrates. In the present study, we find that the rates of superoxide production (measured indirectly as H,02) with glutamate + succinate (similar to 1100 pmol of H2O2 center dot min(-1) mg(-1)) were unexpectedly much higher than with succinate (similar to 400 pmol of H2O2 center dot min(-1) . mg(-1)) or glutamate (similar to 80 pmol of H2O2 center dot min(-1) . mg(-1)) alone. Superoxide production with glutamate + succinate remained high even at low substrate concentrations (< 1 mM), was decreased by rotenone and was completely eliminated by FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), indicating that it must in large part originate from reverse-electron transfer through complex I. Similar results were obtained when glutamate was replaced with pyruvate, a-ketoglutarate or palmitoyl carnitine. In contrast, superoxide production was consistently lowered by the addition of malate (malate + succinate - 30 pmol of H2O2 - min(-1) - mg(-1)). We propose that the inhibitory action of malate on superoxide production can be explained by oxaloacetate inhibition of complex II. In summary, the present results indicate that reverse-electron transfer-mediated superoxide production can occur under physiologically realistic substrate conditions and suggest that oxaloacetate inhibition of complex 11 may be an adaptive mechanism to minimize this.