LAUSR

Abstract The permeabilization of mitochondrial membranes via permeability transition pore opening or by the pore‐forming peptide alamethicin causes a flash of superoxide anion (SA) and hydrogen peroxide production and the inhibition of matrix aconitase. It was shown using the SA probe 3,7‐dihydro‐2‐methyl‐6‐(4‐methoxyphenyl)imidazol[1,2‐a]pyrazine‐3‐one (MCLA) that the substrates of NAD‐dependent dehydrogenases, inhibitors of the respiratory chain, and NAD(P)H at millimolar concentrations suppressed or delayed SA flashes. In the presence of added NADH and NADPH, SA flashes were observed only after considerable oxidation of pyridine nucleotides. The production of SA was maximal at NADPH and NADH redox potentials from −315 to −295 mV and from −325 to −270 mV, respectively, depending on NAD(P)H concentration. SA generation supported by NADPH was severalfold greater than that supported by NADH. In intact mitochondria, NADPH‐ and NADH‐dependent SA generation was negligible. Respiratory substrates at physiological or lower concentrations were incapable of suppressing the NADPH‐supported SA flash. These data indicate that, in conditions close to pathophysiological, matrix NADPH oxidoreductase(s), presumably, an adrenodoxin reductase in complex with adrenodoxin, can essentially contribute to SA flashes associated with transient or irreversible permeability transition pore opening or membrane permeabilization by another mechanism. Graphical abstract Figure. No Caption available. HighlightsPermeabilization of mitochondrial membranes causes a superoxide (SA) flash.Internal (matrix) NAD(P)H oxidoreductases make major contribution to SA flash.Considerable positive shift in NAD(P)H redox potential triggers SA flash.In permeabilized mitochondria external NADPH supports stronger SA flash than NADH.Depletion of respiratory substrates facilitates NAD(P)H‐dependent SA flash.