Ilicicolin Inhibition and Binding at Center N of the Dimeric Cytochrome bc1 Complex Reveal Electron Transfer and Regulatory Interactions between Monomers.
We have determined the kinetics of ilicicolin binding and dissociation at center N of the yeast bc(1) complex and its effect on the reduction of cytochrome b with center P blocked. The addition of ilicicolin to the oxidized complex resulted in a non-linear inhibition of the extent of cytochrome b reduction by quinol together with a shift of the reduced b(H) heme spectrum, indicating electron transfer between monomers. The possibility of a fast exchange of ilicicolin between center N sites was excluded in two ways. First, kinetic modeling showed that fast movement of an inhibitor between monomers would result in a linear inhibition of the extent of cytochrome b reduction through center N. Second, we determined a very slow dissociation rate for ilicicolin (k = 1.2 x 10(-3) s(-1)) as calculated from its displacement by antimycin. Ilicicolin binding to the reduced bc(1) complex occurred in a single phase (k(on) = 1.5-1.7 x 10(5) m(-1) s(-1)) except in the presence of stigmatellin, where a second slower binding phase comprising approximately 50% of the spectral change was observed. This second kinetic event was weakly dependent on ilicicolin concentration, which suggests that binding of ilicicolin to one center N in the dimer transmits a slow (k = 2-3 s(-1)) conformational change that allows binding of the inhibitor in the other monomer. These results, together with the evidence for intermonomeric electron transfer, provide further support for a dimeric model of regulatory interactions between center P and center N sites in the bc(1) complex.
Covian
Raul
R
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Trumpower
Bernard L
BL
D001547:Benzaldehydes / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D001665:Binding Sites / Q000502:physiology; D019281:Dimerization; D004579:Electron Transport / Q000502:physiology; D014450:Electron Transport Complex III / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D006873:Hydroquinones / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D011090:Polyenes / Q000737:chemistry; D020836:Protein Structure, Quaternary / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D001547:Benzaldehydes; D006873:Hydroquinones; D011090:Polyenes; D006418:Heme; C041573:stigmatellin; D014450:Electron Transport Complex III