We have determined the kinetics of ilicicolin binding and dissociation at center N of the yeast bc1 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 bH 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 × 10-3 s-1) as calculated from its displacement by antimycin. Ilicicolin binding to the reduced bc1 complex occurred in a single phase (kon = 1.5–1.7 × 105 m-1 s-1) except in the presence of stigmatellin, where a second slower binding phase comprising ∼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 bc1 complex
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