Cytochrome b5 reductase is the component from neuronal synaptic plasma membrane vesicles that generates superoxide anion upon stimulation by cytochrome c

Abstract

In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b₅ reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b₅ reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b₅ reductase was measured. Complex formation between both proteins suggests that cytochrome b₅ reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochrome b₅ reductase upon complex formation with cytochrome c and suggest a major role of this enzyme as an anti-apoptotic protein during cell death.

Keywords: Cb(5)R, Cytochrome b(5) reductase; Cytochrome b(5) reductase; Cytochrome c; DHE, Dihydroethidium; DTPA, Diethylenetriaminepentaacetic acid; E(+), Ethidium; FAD, Flavin adenine dinucleotide; NADH oxidase; NADH, Reduced nicotinamide adenine dinucleotide; NBT, Nitroblue tetrazolium nitroblue tetrazolium; Neurons; SOD, Superoxide dismutase; SPMV, Synaptic plasma membrane vesicles; Superoxide anion; TB, Terrific Broth terrific Broth; XA, Xanthine xanthine; XO, Xanthine oxidase.