EPR studies on the iron-sulfur centers of dpnh dehydrogenase during the redox cycle of the enzyme

Preparations of the inner mitochondrial membrane, upon treatment with DPNH in the aerobic state, undergo a cycle of absorbance changes, which may be monitored at 470-500 mμ. There is rapid initial bleaching, followed by return of the color on exhaustion of DPNH. 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. The EPR signals of four different iron-sulfur centers associated with DPNH dehydrogenase have thus far been resolved at temperatures between 4-20°K. Of these the iron-sulfur center of lowest potential, center 1, is reoxidized by the respiratory chain when DPNH is exhausted, but the high potential center 2 remains reduced. ATP reoxidizes iron-sulfur center 2 and restores full color to the chromophore. It is suggested that the energy for the reoxidation of the chromophore and iron-sulfur center 2 is supplied by coupling site I. On the basis of these and of other observations it is tentatively proposed that coupling site I is directly associated with DPNH 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.