Relation of the Respiratory Chain-Linked Reduced Nicotinamide-Adenine Dinucleotide Dehydrogenase to Energy-Coupling Site

Preparations of submitochondrial particles, upon treatment with reduced nicotinamide-adenine dinucleotide (NADH) in the aerobic state, undergo a cycle of absorbance changes, which may be monitored at the 470- minus 500-m/x wavelength pair. There is a rapid initial bleaching, followed by return of the color on exhaustion of the NADH. In the presence of rotenone or piericidin the cycle is prolonged, the return of color (reoxidation) is inhibited and the extent of reoxidation less than in uninhibited particles. On the basis of prior work the residual bleaching (and most or all of the initial bleaching by NADH) is thought to be due to chromophores associated with NADH dehydrogenase. ATP causes rapid reoxidation of the chromophore which remains permanently reduced in rotenone- and piericidin-blocked preparations. This effect is inhibited by oligomycin and 2, 4-dinitrophenol. While ATP causes a complete reoxidation of the nonheme iron chromophores, it reduces cytochrome b in rotenone-inhibited preparations but this reduction is transient. Reoxidation of the chromophore monitored at 470 — 500 mμ is suggested to be an intraenzymic reversed electron flow, energized by ATP via phosphorylation site 1. Of the various iron-sulfur species associated with NADH dehydrogenase, whose reduction gives rise to characteristic electron paramagnetic resonance signals observed at temperatures between 4 and 20 °K, the low potential iron-sulfur center 1 is reoxidized by the respiratory chain when NADH is exhausted, but the higher potential center 2 remains reduced. ATP causes reoxidation of the iron-sulfur of center 2. On the basis of these and of other observations it is tentatively proposed that coupling site 1 is directly associated with NADH dehydrogenase and is located on the 02 side of iron-sulfur center 1 and the substrate side of both center 2 and the specific binding sites of rotenone and piericidin.