Energy conservation by the plant mitochondrial cyanide-insensitive oxidase. Some additional evidence

Abstract

Several measures of energy conservation, namely ADP/O ratio, P/O ratio, ATP/O ratio and phosphorylation detected by continuous assay with purified firefly luciferase and luciferin, all show phosphorylation can occur with mung-bean mitochondria at cyanide concentrations sufficient to inhibit the cytochrome oxidase system. Phosphorylation in the presence of cyanide is uncoupler- oligomycin- and salicylhydroxamate-sensitive. The participation of phosphorylation site 1 is excluded, phosphorylation being attributable to a single phosphorylation site associated with the cyanide-insensitive oxidase. The cyanide-insensitive oxidase has also been shown to support a variety of other energy-linked functions, namely, Ca2+ uptake, reversed electron transport and the maintenance of a membrane potential detected by the dye probes 8-anilinonaphthalene-1-sulphonate and safranine. High concentrations of cyanide have uncoupler-like activity, decreasing the ADP/O ratio and the t 1/2 for the decay of a pH pulse through the the mitochondrial membrane. This uncoupler-like effect is most marked with aged mitochondria. The observations of energy conservation attributable to the cyanide-insensitive oxidase are compared with other reports where it is concluded that the alternative oxidase is uncoupled.

Cyanide‐resistant respiration: A non‐phosphorylating electron transport pathway acting as an energy overflow

“…ubiquinone) via the alternative pathway is neither coupled with phosphorylation, nor with the generation of a membrane potential or reverse electron transport in the cytochrome path (Laties 1982). However, Wilson (1980) claimed that the cyanide-insensitive oxidase in mitochondria from mung bean (Phaseolus aureus) can support a variety of energy-linked functions, including phosphorylation at one site. Wilson explained the discrepancy between his and other workers' results by the "uncoupler-like activity" of cyanide, and stated that studies of energy conservation in the alternative pathway should be carried out at low cyanide concentrations.…”

Effects of amiprophosmethyl on massive and limited calcium loading of maize mitochondria

“…6). We suggest that this may be due to endogenous oxidizable substrates, which may support Ca2+ transport by the cyanide-insensitive pathway (Wilson, 1980), which itself is not inhibited by rotenone (Moore & Rich, 1980). Addition of succinate increased this Ca2+ accumulation to 16 nmol of Ca2+/mg of mitochondrial protein (see above).…”

The switching of electron flux from the cyanide-insensitive oxidase to the cytochrome pathway in mung-bean (Phaseolus aureus L.) mitochondria

“…Mung-bean mitochondria prepared as described above have about 30-40 % of their succinate-supported 02-Vol. 249 uptake rate insensitive to cyanide (see Wilson, 1980) and have ADP/0 ratios indicative ofcoupled and intact plant mitochondria. In the presence of succinate and KCN, 02 uptake can be used to measure the alternativepathway activity, whereas electron flux using the cytochrome b-c segment of the conventional respiratory chain is observed by spectrophotometric monitoring of ferricyanide reduction.…”

“…The observation that electrons can be switched from alternative to cytochrome oxidase and vice versa is perhaps not surprising; both pathways probably derive their electrons from a common ubiquinone pool, and modern control theory (Kacser & Burns, 1973;Kacser, 1983) predicts that, in a branched pathway, restriction of the function of any enzyme will modify the flux through all parts of the system. Reversible switching of the electron flux, the single phosphorylation site that can be observed on the alternative-oxidase pathway between the branch point and 02 (ADP/0 close to 1 for quinone to oxygen in the presence of cyanide) (Wilson, 1970(Wilson, , 1978(Wilson, , 1980 compared with two sites on the corresponding cytochrome pathway, and the branched-dehydrogenase segment of the plant mitochondrial electron-transport system, all indicate a complex and metabolically flexible system which must possess an equally complex and as yet ill-understood regulatory system.…”