Abstract
Bovine heart bc1 complex is reversibly inhibited by zinc ions with an inhibition constant KI of 10(-7) M at pH > or = 7.0. Binding of zinc is at least a factor of 10 tighter than binding of any other metal ion tested. Essentially complete inhibition of ubihydroquinone:cytochrome c oxidoreductase activity is observed at concentrations of [Zn2+] > 5 microM. Zinc does not affect the Km for the substrates, ubihydroquinone or cytochrome c, but zinc inhibits reduction of the cytochromes by ubihydroquinone through the QP center. A radioactive binding assay using 65Zn revealed one high affinity binding site per bc1 complex with KD < or = 10(-7) M at pH = 7.0 and 3-4 additional low affinity binding sites (KD > 2 x 10(-6) M). Zinc binding does not depend on the redox state of the high potential chain (iron-sulfur protein and cytochrome c1). Zinc binds 3 times tighter to Fe-S-depleted bc1 complex indicating that the zinc binding site is not on the "Rieske" iron-sulfur protein in contrast to a recent report by Lorusso et al. (Lorusso, M., Cocco, T., Sardanella, A.M., Minuto, M., Bonomi, F., and Papa, S. (1991) Eur. J. Biochem. 197, 555-561). Zinc binds to a site which has the same affinity for zinc as for protons. We conclude that the zinc binding site is close to a protonatable group of the bc1 complex with pKa = 7.2 which has not been identified previously. We propose that this group is part of the proton channel at the hydroquinone oxidation center of the bc1 complex.
Highlights
Inhibitors have been indispensable tools for elucidating the reactions of the ubiquitous bc1 complexes
Bovine heart mitochondria were prepared according to Smith [7]. bc1 complex was prepared as described by Schagger et al [8] with the following modification: the buffer for the final Sepharose CL-6B gel filtration column was 0.05% Triton X-100, 100 mM NaNO3, 20 mM Pipes,1 pH ϭ 7.2
Metal Ion Inhibition of Bovine Heart Mitochondria—Inhibition of succinate:O2 respiration was measured in uncoupled mitochondria washed with Chelex-purified buffer
Summary
Inhibitors have been indispensable tools for elucidating the reactions of the ubiquitous bc complexes (for a review, see Ref. 1). Almost all of these inhibitors are aromatic organic compounds where at least part of the structure has some structural relationship to the substrate, ubiquinone. The “Q-cycle” mechanism has been established for the sequence of electron and proton transfers In the light of this body of information, we have reinvestigated inhibition by zinc to obtain information about the specific interaction between zinc ions and the bc complex and to use this information to get insight into mechanistic details of the electron and proton transfer reactions
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