Abstract
We have obtained evidence for electron transfer between cytochrome b subunits of the yeast bc(1) complex dimer by analyzing pre-steady state reduction of cytochrome b in the presence of center P inhibitors. The kinetics and extent of cytochrome b reduced by quinol in the presence of variable concentrations of antimycin decreased non-linearly and could only be fitted to a model in which electrons entering through one center N can equilibrate between the two cytochrome b subunits of the bc(1) complex dimer. The b(H) heme absorbance in a bc(1) complex inhibited at center P and preincubated with substoichiometric concentrations of antimycin showed a red shift upon the addition of substrate, which indicates that electrons from the uninhibited center N in one monomer are able to reach the b(H) heme at the antimycin-blocked site in the other. The extent of cytochrome b reduction by variable concentrations of menaquinol could only be fitted to a kinetic model that assumes electron equilibration between center N sites in the dimer. Kinetic simulations showed that non-rate-limiting electron equilibration between the two b(H) hemes in the dimer through the two b(L) hemes is possible upon reduction through one center N despite the thermodynamically unfavorable b(H) to b(L) electron transfer step. We propose that electron transfer between cytochrome b subunits minimizes the formation of semiquinone-ferrocytochrome b(H) complexes at center N and favors ubiquinol oxidation at center P by increasing the amount of oxidized cytochrome b.
Highlights
Sources [3,4,5,6], and this stabilization helps to explain why superoxide formation at center N is virtually non-existent when center P is blocked with stigmatellin [7, 8]
The kinetics and extent of cytochrome b reduced by quinol in the presence of variable concentrations of antimycin decreased non-linearly and could only be fitted to a model in which electrons entering through one center N can equilibrate between the two cytochrome b subunits of the bc1 complex dimer
The bH heme absorbance in a bc1 complex inhibited at center P and preincubated with substoichiometric concentrations of antimycin showed a red shift upon the addition of substrate, which indicates that electrons from the uninhibited center N in one monomer are able to reach the bH heme at the antimycin-blocked site in the other
Summary
Q, quinone; QH2, quinol; SQ, semiquinone; DBH2, decylubiquinol (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinol); MQH2, menaquinol (2,3-dimethyl-1,4-naphthoquinol); MQ, menaquinone; MSQ, menasemiquinone; E, enzyme; I, inhibitor. According to electron tunneling calculations, such distance should allow electron transfer at a rate in the order of 104-105 sϪ1 [14] Before this structural information was available, kinetic and spectroscopic evidence was used to propose direct electron equilibration between the two center N sites of the dimer [15, 16]. Such equilibration was assumed to occur by direct electron transfer between the Q molecules bound at the two center N sites and not by electron communication between the bL hemes. We suggest that a possible function of this intermonomer electron communication is to use the stabilization of SQ at center N to maintain the bH hemes in the oxidized state, ensuring a maximal rate of QH2 oxidation at center P
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