Wurster's blue mediated oxidation of NADH and phosphorylation in mitochondria

Abstract

The slow oxidation of NADH 2 2 Abbreviations used: NADH, reduced nicotinamide adenine dinucleotide; TMPD, N,N,N′,N′-tetramethyl- p-phenylenediamine; WB, Wurster's Blue, a semiquindiimine radical of TMPD; Seconal, 5-allyl-5(1-methylbutyl)-barbituric acid; HOQNO, 2- N-nonylhydroxyquinoline- N-oxide. in liver mitochondria is accelerated by Wurster's blue (WB), a semiquindiimine radical of N,N,N',N'-tetramethyl- p-phenylenedia-mine (TMPD). The inhibition of NADH oxidation by Rotenone, Seconal, or antimycin A is bypassed through WB; thus the NADH-WB system acts as an electron feeder to the terminal region of the respiratory chain. The oxidation of NADH-WB by liver mitochondria yields an ADP O ratio of nearly 2 when the concentration of WB added is low. This ratio is further decreased to about 1 in the presence of Rotenone, antimycin A, or high concentrations of WB, i.e., the conditions which prevent the oxidation of endogenous substrates and eliminate the first two phosphorylation sites. The oxidation of NADH-WB by liver mitochondria or cardiac submitochondrial particles, in the presence or absence of Rotenone, results in the reduction of all the cytochromes. In the presence of antimycin A, the addition of NADH-WB causes an oxidation of cytochrome b, but a reduction of cytochromes c 1 + c and a + a 3. This oxidation of cytochrome b is explained by the occurrence of a WB-mediated bypass of electron transfer over the antimycin-sensitive site. In the absence of antimycin A, the reduction of cytochrome b that occurs is the result of a reversed electron flow associated with the oxidation of NADH-WB system. This reduction of cytochrome b can be prevented under conditions in which the reversed electron flow is blocked, such as by addition of cyanide plus Rotenone. However, the cytochrome b reduction can also be induced in this inhibited system if ATP is used as an energy source to drive the reversed electron flow. These results have been interpreted with respect to the egresses and entries of electrons along the respiratory chain.