Susanne Jünemann

ON THE MECHANISM OF QUINOL OXIDATION IN THE BC1 COMPLEX

The question of whether significant levels of a semiquinone can be generated in the Qo site of thebc 1 complex under conditions of oxidant-induced reduction is relevant to the mechanism of bifurcation of electron transfer in this site. It has already been reported that beef heart submitochondrial particles under such conditions exhibit an EPR-detectable semiquinone, which is distinct from Q⨪i and which was attributed to a semiquinone in the Qo site (de Vries, S., Albracht, S. P. J., Berden, J. A., and Slater, E. C. (1981) J. Biol. Chem. 256, 11996–11998). However, we show here that this signal, which can be generated to a level of around 0.1 perbc 1 monomer, is insensitive to the Qo site inhibitors myxothiazol, E-β-methoxyacrylate-stilbene, and stigmatellin, indicating that it does not arise from a Q⨪o species. Based on sensitivities to inhibitors of other Q sites, up to 60% of the signal may arise from semiquinones of complexes I and II. We further show that the iron-sulfur center remains EPR silent under oxidant-induced reduction conditions. Overall, the results indicate that, under conditions of oxidant-induced reduction, the Qo site is occupied primarily by quinol with the iron-sulfur center oxidized, or, possibly, by an antiferromagnetically coupled semiquinone/reduced iron-sulfur center pair, which are EPR silent. This is discussed in relation to proposed mechanisms of quinol oxidation in the Qo site, and we describe a minimal intermediate-controlled bifurcation model based on rate constants by which bifurcated electron transfer at the Qo site might occur.

PROTONMOTIVE MECHANISM OF HEME-COPPER OXIDASES

The mechanism of coupling of proton and electron transfer in oxidases is reviewed and related to the structural information that is now available. A “glutamate trap” mechanism for proton/electron coupling is described.

Antimycin inhibition of the cytochrome bd complex from Azotobacter vinelandii indicates the presence of a branched electron transfer pathway for the oxidation of ubiquinol

Antimycin A and UHBDT inhibit the activity of the purified cytochrome bd complex from Azotobacter vinelandii. Inhibition of activity is non‐competitive and antimycin A binding induces a shift to the red in the spectrum of a b‐type haem. No inhibitory effects were seen with myxothiazol. Steady‐state experiments indicate that the site of inhibition for antimycin A lies on the low‐potential side of haem b 558. In the presence of antimycin A at concentrations sufficient to inhibit respiration, some direct electron transfer from ubiquinol‐1 to haem b 595 and haem d still occurs. The results are consistent with a branched electron transfer pathway from ubiquinol to the oxygen reduction site.

Coupling of Ion and Charge Movements: From Peroxidases to Protonmotive Oxidases

The energy cost of introduction of charges into regions of low dielectric strength in proteins can be reduced by associated binding of a proton to appropriately placed residues. Such protonations can be important in peroxidases and other soluble proteins, and are likely to be central to the protonmotive mechanism of oxidases. Three residues in subunit I of cytochrome oxidase that are likely to interfere with such protonations have been examined by mutagenesis, and the data are discussed in the light of the known crystal structures.