Succinate oxidation by coupled potato tuber mitochondria followed by rapid scan spectrometry

Abstract

Oxidation of succinate by potato tuber mitochondria has been investigated from aerobiosis to complete anuerobiosis. Difference spectra of the various steps were recorded by a rapid scan spectrometer delivering averaged spectra every 3 s in the range 380 to 630 mm. The transitions between state 3 and 4 resulted in large redox changes, essentially for the b cytochromes, and in significant changes in the spectral baseline (light scattering). At anaerobiosis the cytochromes c, c1 and a were reduced while cytochrome a, remained oxidized. – Addition of uncouplers in aerobiosis induced oxidation of the b cytochromes, and when anaerobiosis occurred cytochromes c, c1a and a3 were reduced simultaneously. When uncouplers were added in anaerobiosis a partial oxidation of the b cytochromes and the reduction of cytochrome a3 were observed. These results are interpreted as the building up of a membrane potential, maximal in state 4 and stable after anaerobiosis. The cytochromes buried in the membrane equilibrate with the membrane potential, and their redox states are sensitive to the changes. Variations of membrane potential also induce changes in the light scattering by the mitochondrial membrane.

Rapid scan spectrometry: oxidation of substrates by coupled potato mitrochondria

“…A fast reduction of a cytochromes (445-455) ntn is observed during this time, atid afterwards it continues slowly: this slow process corresponds to the reduction of cytochroine 03. Indeed, in coupled mitochondria only cytochronne a is rapidly reduced at anaerobiosis (Ducet 1980). The (430-410) nm trace shows a lag of 3 s (on spectrum) before a rapid increase in reduction (reduction before anaerobiosis is the one occurring in state 4 as illustrated by Figure 3B, c).…”

“…A study of the oxidation of succinate in energized and Materials deenergized states has been reported (Ducet 1980). Potato tuber, Solum tuberosum L. cv.…”

“…tential, maxitnal in state 4 and collapsed by uncouplers, and suggested that the redox states of the b and a cytochromes were dependent on this membrane potential. Methods The changes in the shape of the spectra as weJI as in Mitochondria were prepared and assayed as previously their base line level were also related to membrane po-described (Ducet 1980). tential variations.…”

“…For the three investigated substrate oxidations, addition of 1999 in anaerobiosis induced a partial oxidation of the b cytochromes and a further reduction of cytochrotne a 3 (Ducet 1980). This is illustrated by traces (430-410) nm and (445-455) nm of Figure 5, The oxidation of ft cytochromes took place 3 s before cytochrome ^3 reduction but was synchronous with a small flavoprotein oxidation |trace (460-410) nm].…”

“…The apparent reactivity of only the t cytochromes during aerobiosis could indicate either a crossover point (Chance and Williams 1956), or a gap between b and c cytochromes due to intermediate factors (Trumpower 1976) or reverse electron transfer in state 4 (Rieske 1976). As b cytochromes are integrated in the inner membrane, changes of membrane potential could also influence their apparent redox state (Ducet 1980), since the degree of reduction of the b cytochromes depends on the substrate oxidized and on the energized or deenergized conditions of mitochondria (Wikstrom 1973).…”