Proton translocation linked to the activity of the bi-trans-membrane electron transport chain

Recently we have proposed and presented evidence suggesting the existence of a "bi-trans-membrane" electron transport chain, located at the contact sites between outer and inner mitochondrial membranes, which can be utilized to promote either the oxidation of exogenous NADH in the presence of catalytic amounts of added cytochrome c or the reduction of exogenous cytochrome c supported by the oxidation of respiratory substrates present inside the mitochondria. Here we show that the oxidation of exogenous NADH is accompanied by a net alkalinization of the incubation medium preceded by a transient acidification phase. In oxygen-pulse experiments, the alcohol oxidation (induced by the addition of alcohol dehydrogenase) was used to mimic a cytosolic source of reducing equivalents. Oxygen pulses promote an acidification-alkalinization proton cycle which is insensitive to antimycin and myxothiazol inhibitory effect, is stimulated by valinomycin, inhibited by trypsin-aprotinin complex, abolished by the protonophore carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP), and is absent or at least inverted (alkalinization-acidification cycle) in broken mitochondria. The oxidation of cytosolic substrates, mediated by the bi-trans-membrane electron transport chain, does not involve endogenous cytochrome c and is associated with a vectorial proton translocation from the inside to the outside of the mitochondria. In the out-->in electron transport pathway the components involved appear to be cytosolic reduced substrates-->NADH produced by cytosolic dehydrogenases activity-->NADH-cytochrome b5 oxidoreductase complex leaning out the external side of the external membrane-->exogenous cytochrome c-->cytochrome oxidase of contact sites-->molecular oxygen. The possible components of the in-->out pathway are matrix respiratory substrates-->primary dehydrogenases of the matrix-->Complexes I, II, and III of the respiratory chain present in the inner membrane-->NADH-cytochrome b5 oxidoreductase system of the external membrane-->exogenous cytochrome c-->additional cytosolic electron acceptors or, alternatively, cytochrome oxidase of contact sites. The two pathways can be considered a bi-trans-membrane electron channeling system which, at the level of bridges set up by the contact points between the outer and the inner mitochondrial membrane, may represent a link between the redox processes occurring inside with those present outside the mitochondrion.