Kinetic of superoxide formation by respiratory chain NADH-dehydrogenase of bovine heart mitochondria

Formation of superoxide anions (O₂-) by bovine heart NADH-dehydrogenase preparation (Complex I) supported by an NADH- or NADPH-generating system was studied kinetically. Both NADH- and NADPH-dependent superoxide-forming activities of Complex I were biphasic in double reciprocal plots. The NADHdependent reaction had two sets of kinetic parameters: One K_m value for NADH was 10 times higher than the other one, and the V_max of the reaction with high K_m was about 4 times higher than that of the reaction with low K_m. Similar V_max values were obtained for the NADPH-dependent reactions but the K_m values were a thousand times higher than those of the NADH-dependent reactions. The plots of the NADH-dependent activity of rotenone-treated submitochondrial particles were also biphasic. The double reciprocal plots of NADH- and NADPH-dependent 2,6-dichlorophenolindophenol (DCIP) reductase activities of Complex I were linear and the plots of the superoxide-forming activities against the DCIP reductase activities were biphasic. These results indicate that the biphasic double reciprocal plots of the superoxide-forming activities against reduced coenzymes are not caused by interaction between reduced coenzymes and the NADH-dehydrogenase but are due to the presence of at least two superoxide-forming sites in the respiratory chain NADH-dehydrogenase.