Baker's yeast flavocytochrome b2. A mechanistic study of the dehydrohalogenation reaction

It has been shown that reduced flavocytochrome b2 not only catalyzes reduction of bromopyruvate [P. Urban, P.M. Alliel and F. Lederer (1983) Eur. J. Biochem. 134, 275-281] but also transforms it into pyruvate in a reductive elimination process. The dehydrohalogenation reaction also takes place when oxidized enzyme acts on bromolactate, but the reaction is more difficult to observe under these conditions because of its low efficiency compared to the normal oxidative process. The maximal rates of pyruvate production from bromopyruvate and chloropyruvate differ by a factor of less than 10, whereas elimination from fluoropyruvate cannot be detected. These results support a mechanism in which the dehydrohalogenation reaction takes place from a carbanion intermediate of the normal reductive-oxidative pathway.     

Coupling between redox and acid-base energy by cytochrome b-564 in Baker's yeast mitochondria

Baker's yeast mitochondrial cytochrome b-564 is characterized by exhibiting both a labile pH-independent high-potential form (E'o, pH 7 = + 190 mV) and a stable pH-dependent (pKa = 6.8) low-potential form (E'o, pH 7 = + 70 mV). The different behavior of these two forms of cytochrome b-564 versus pH seems to be a decisive factor for transduction of redox energy into acid-base energy in oxidative phosphorylation site 2. Deenergizing treatments, such as ADP plus Pi, result in the conversion of all the mitochondrial cytochrome b-564 into its low-potential form, whereas energization with ATP specifically transforms the cytochrome into its high-potential form, the ATP effect being neutralized by the ATPase inhibitor oligomycin and by the uncoupler FCCP. Accordingly, a minimal model for coupling between redox energy and acid-base energy through an electronically energized and protonated ferricytochrome b-564 intermediate is proposed. The energy-transducing properties of mitochondrial cytochrome b-564 seems to be shared by chloroplast cytochrome b-559.     

Region of cytochrome c interacting with yeast cytochrome b2: determination with singly modified carboxydinitrophenyl cytochromes c

The association and reduction reactions of ten different 4-carboxy-2,6-dinitrophenyl (CDNP) horse heart cytochromes c, singly modified at lysines 8, 13, 27, 39, 60, 72, 73, 86, 87, and 99, with Saccharomyces cerevisiae cytochrome b2 were studied to determine the region of cytochrome c interacting with cytochrome b2. In the presence of higher ratios of free cytochrome c to cytochrome b2, native cytochrome c, and the CDNP-lysine 39, 60, and 99 derivatives associated with cytochrome b2 with a binding stoichiometry close to 2:1, while CDNP-cytochromes c modified at lysines 8, 13, 27, 72, 73, 86, and 87 formed only 1:1 complexes. In the presence of lower ratios of free cytochrome c, modifications of lysines 8, 27, 86, and 87 had more inhibitory effects on the association of cytochrome c with cytochrome b2 than modifications of lysines 13, 39, 60, 72, 73, and 99. This tendency was similar to that on removal of free cytochrome c, except in the case of CDNP-lysine 13 and 73 derivatives. The rate of reduction of cytochrome c by cytochrome b2 was decreased by carboxydinitrophenylation of lysines 8, 13, 27, 72, 73, 86, and 87. In contrast, the rate of reduction of cytochrome c was not affected by modifications of lysines 39, 60, and 99. Since lysines 8, 13, 27, 72, 73, 86, and 87 are located on the front surface and lysines 39, 60, and 99 on the back side, and since different effects of modifying lysine residues located on the front surface may be interpreted in terms of effects on the complementary interaction of cytochrome c and cytochrome b2, these results indicate that the region of cytochrome c interacting with cytochrome b2 is located on the front surface of the cytochrome c molecule containing the exposed heme edge.