General Nature of “Wurster's Blue Shunts” in the Respiratory Chain

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Abstract Wurster's blue, a semiquindiimine radical of N,N,N',N'-tetramethyl-p-phenylenediamine, acts as an artificial electron carrier in the oxidation of substrates by the respiratory chain. The effect of this compound on the oxidation of various substrates has been studied at three levels of organization: submitochondrial particles, mitochondria, and intact tissue culture cells. In cardiac submitochondrial particles (the Keilin-Hartree preparation), as judged by the rate of oxygen consumption, the inhibition of NADH and succinate oxidation by various respiratory inhibitors was bypassed through Wurster's blue. The typical inhibitors were antimycin A, hydroxyquinoline-N-oxide, triflurothenoylacetone, rotenone, Amytal, and Seconal. In liver mitochondria, studied at States 3 and 4, the observation was the same whether β-hydroxybutyrate or succinate was used as a substrate. The phosphorylation was partially restored by Wurster's blue in the inhibited systems of mitochondria. The P:O ratios of about 1 and 2 resulted for oxidation of succinate-Wurster's blue and β-hydroxybutyrate-Wurster's blue, respectively, when antimycin A was present. However, the P:O ratio was 1 in the presence of rotenone, Amytal, Seconal, or antimycin A plus Seconal for the β-hydroxybutyrate-Wurster's blue system. In systems of tissue culture cells, the inhibition of endogenous respiration was reversed by Wurster's blue. Cellular ATP content decreased considerably in the presence of Wurster's blue, but never reached zero. The viability of cells was practically not affected by Wurster's blue during a 4-hour incubation. The oxidation-reduction states of cytochromes were followed with respect to the interaction of substrates plus Wurster's blue with the respiratory chain in the presence and absence of various inhibitors. An energy-dependent reduction of cytochrome b via the reversed electron transport utilizing the reducing equivalents from Wurster's blue-substrate systems was shown. The results of the experiments on respiration and phosphorylation together with spectrophotometric evidence indicate the general nature of Wurster's blue shunts in the respiratory chain. The results are also interpreted with respect to the egresses and entries of electrons of the shunts.