Reduction kinetics of cytochromes b

Abstract 1. 1. Durohydroquinone (H 2 DQ), a specific reductant for the second phosphorylation site, has been used in the study of the fast reduction kinetics of cytochromes b . 2. 2. In ubiquinone-extracted mitochondria, H 2 DQ reduces cytochrome(s) b in a fast reaction, the rate and extent of which decreases upon reincorporation of ubiquinone. 3. 3. In cytochrome c -extracted mitochondria, H 2 DQ reduces both cytochromes b . After ATP addition, with cytochrome b T reduced by endogenous substrate, H 2 DQ reduces cytochrome b K at a slow rate whereas ubiquinone readily accepts reducing equivalents. In the presence of uncoupler, H 2 DQ reduces cytochrome b K at a faster rate while ubiquinone behaves as a less efficient electron acceptor. Under these conditions, cytochrome b T is not reduced by H 2 DQ. 4. 4. Cytochrome c 1 reduction can be resolved free of cytochrome c interference in cytochrome c -extracted mitochondria. Cytochrome c 1 reduction is 4 times faster in the presence of uncoupler than in the presence of ATP. 5. 5. Addition of cytochrome c to cytochrome c -extracted mitochondria stimulates the rate of cytochromes b reduction. 6. 6. In intact mitochondria, ATP addition causes 20 % reduction of cytochrome b T and speeds up the initial rate of cytochromes b reduction by a factor of 10. 7. 7. Addition of antimycin A to intact mitochondria results in 80–100 % reduction of cytochrome b K by endogenous substrate. ATP added to antimycin A-inhibited mitochondria produces oxidation of cytochrome b K and simultaneous and variable reduction of cytochrome b T . This latter effect is reverted by the addition of an uncoupler.