Zinc Ions Inhibit the Qp Center of Bovine Heart Mitochondrial bc1 Complex by Blocking a Protonatable Group

Abstract

. 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 bc 1 complex with pK a ‫؍‬ 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 bc 1 complex.Inhibitors have been indispensable tools for elucidating the reactions of the ubiquitous bc 1 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 single notable exception are zinc ions which have first been reported by Skulachev et al. (2) to inhibit mitochondrial respiration in micromolar concentrations. Subsequent studies (3-5) have established that the primary site of zinc inhibition in bovine heart mitochondria is the bc 1 complex.The knowledge about the bc 1 complex has largely increased over the past 20 years, including the structure of the redox centers and the peptide composition of the mammalian 11-subunit complex. The "Q-cycle" mechanism has been established for the sequence of electron and proton transfers (see Ref. 6). 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 1 complex and to use this information to get insight into mechanistic details of the electron and proton transfer reactions. MATERIALS AND METHODSMetal salts were obtained either as chloride or as nitrate salts from Fluka. Cytochrome c (horse heart) was from Sigma, prepared without trichloroacetic acid.Bovine heart mitochondria were prepared according to Smith (7). bc 1 complex was prepared as described by Schä gger 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 NaNO 3 , 20 mM Pipes, 1 pH ϭ 7.2 (no NaN 3 ). Fe-S-deficient bc 1 complex ϩ antimycin was prepared following the procedure given by Schä gger et al. (9). After complete elution of the iron-sulfur protein, Fe-S-deficient bc 1 complex ϩ antimycin was eluted with 0.05% Triton X-100, 150 mM sodium phosphate, pH 7.2, and applied to a gel filtration column (Sepharose CL-6B, Pharmacia) equilibrated with 0.05% Triton X-100, 100 mM NaNO 3 , 20 mM Pipes, pH 7.2, and run with the same buffer. The fractions containing Fe-S-deficient bc 1 complex ϩ antimycin were mixed with 10% (w/v) glycerol and frozen in liquid nitrogen.Buffer for kinetic measurements was prepared as follows: 200 mM sucrose, 50 mM Pipes (from a partially neutralized stock solution), and 1 mM NaN 3 were dissolved in tridistilled water. The solution was purified over a Chelex 100 ion exchange resin column (analytical grade, Bio-Rad). 20 mM HNO 3 , 1 mM Ca(NO 3 ) 2 , and 0.024% (0.5 mM) dodecyl maltoside (Boehringer) were added, and the pH was adjusted with 2 M NaOH (purified over Chelex 100 and stored in a plastic bottle). Calcium was ...

Terminal oxidation and the effects of zinc in prostate versus liver mitochondria

“…Skulachev et al (1967) found that μM free zinc ions inhibited electron transfer between cytochrome b and c 1 of complex III and had no effect on the cytochrome c oxidase; and our results are in general agreement with those observations. Similar observations were reported by studies in bovine heart mitochondria (Nicholls and Malviya, 1968;Kleiner and von Jagow, 1972;Lorusso et al, 1991;Link and von Jagow, 1995). Link and von Jagow (1995) proposed that zinc inhibits complex III by interfering with proton transfer reactions at the Q p center of the bc 1 complex.…”

“…Similar observations were reported by studies in bovine heart mitochondria (Nicholls and Malviya, 1968;Kleiner and von Jagow, 1972;Lorusso et al, 1991;Link and von Jagow, 1995). Link and von Jagow (1995) proposed that zinc inhibits complex III by interfering with proton transfer reactions at the Q p center of the bc 1 complex. However, all these studies involved effects of free Zn + + ions, which do not exist in the intracellular environment.…”

“…The inhibition of both succinate and NADH oxidation by prostate and liver mitochondria leading to cytochrome c reduction strongly supports the likely inhibition of complex III by zinc. However, Link and von Jagow (1995) determined that the bc1 zinc-binding sites have binding affinities equivalent to log K f ~ 6-7. Seemingly, this would be inconsistent with the effective inhibition obtained with ZnCysteine (log K f~ 10).…”

“…Several studies with mammalian cells have reported that zinc ions inhibit mitochondrial respiration and terminal oxidation (Hunter and Ford, 1955;Skulachev et al, 1967;Nicholls and Malviya, 1968;Kleiner and von Jagow, 1972;Lorusso et al, 1991;Link and von Jagow, 1995). However all these studies employed μM-mM concentrations of free Zn + + ions; i.e.…”

Modulation of mitochondrial function by endogenous Zn 2+ pools

“…In previous studies we have found that, on strong cytosolic loading, Zn 2ϩ is taken up into these organelles, from which it can be subsequently released in a Ca 2ϩ dependent fashion (10,11). Zn 2ϩ accumulation in mitochondria may not be benign, as Zn 2ϩ has been found to interfere with the function of isolated mitochondria (12)(13)(14)(15)(16)(17)(18)(19) and mitochondria are therefore likely to be sites at which this cation induces injurious effects on neurons (20,21).…”