T. V. Vygodina

Zinc ions as cytochrome c oxidase inhibitors: two sites of action

Zinc ions are shown to be an efficient inhibitor of mitochondrial cytochrome c oxidase activity, both in the solubilized and the liposome reconstituted enzyme. The effect of zinc is biphasic. First there occurs rapid interaction of zinc with the enzyme at a site exposed to the aqueous phase corresponding to the mitochondrial matrix. This interaction is fully reversed by EDTA and results in a partial inhibition of the enzyme activity (50–90%,depending on preparation) with an effective Ki of ∼10 µM. The rapid effect of zinc is observed with the solubilized enzyme, it vanishes upon incorporation of cytochrome oxidase in liposomes,and it re-appears when proteoliposomes are supplied with alamethicin that makes the membrane permeable to low molecular weight substances. Zinc presumably blocks the entrance of the D-protonic channel opening into the inner aqueous phase. Second, zinc interacts slowly (tens of minutes, hours) with a site of cytochrome oxidase accessible from the outer aqueous phase bringing about complete inhibition of the enzymatic activity. The slow phase is characterized by high affinity of the inhibitor for the enzyme:full inhibition can be achieved upon incubation of the solubilized oxidase for 24 h with zinc concentration as low as 2 µM. The rate of zinc inhibitory action in the slow phase is proportional to Zn2+ concentration. The slow interaction of zinc with the outer surface of liposome-reconstituted cytochrome oxidase is observed only with the enzyme turning over or in the presence of weak reductants, whereas incubation of zinc with the fully oxidized proteoliposomes does not induce the inhibition. It is shown that zinc ions added to cytochrome oxidase proteoliposomes from the outside inhibit specifically the slow electrogenic phase of proton transfer, coupled to a transition of cytochrome oxidase from the oxo-ferryl to the oxidized state (the F → O step corresponding to transfer of the 4th electron in the catalytic cycle).

Cytochrome oxidase-catalyzed superoxide generation from hydrogen peroxide

Superoxide dismutase is shown to affect spectral changes observed upon cytochroma c oxidase reaction with H2O2, which indicates a possibility of O− 2 radicals being formed in the reaction. Using DMPO as a spin trap, generation of superoxide radicals from H2O2 in the presence of cytochrome oxidase is directly demonstrated. The process is inhibited by cyanide and is not observed with a licat‐denatured enzyme pointing to a speciric reaction in the oxygen‐reducing centre of cytochrome c oxidase. The data support a hypothesis on a catalase cycle catalysed by cylochrome c oxidase in the presence of excess H2O2 (Vygodina and Konstantinov (1988) Ann. NY Acad. Sci., 550, 124‐138);

Time-resolved generation of a membrane potential by ba3 cytochrome c oxidase from Thermus thermophilus Evidence for reduction-induced opening of the binuclear center

ba 3‐type cytochrome c oxidase purified from the thermophilic bacterium Thermus thermophilus has been reconstituted in phospholipid vesicles and laser flash‐induced generation of a membrane potential by the enzyme has been studied in a μs/ms time scale with Ru(II)‐tris‐bipyridyl complex (RuBpy) as a photoreductant. Flash‐induced single electron reduction of the aerobically oxidized ba 3 by RuBpy results in two phases of membrane potential generation by the enzyme with τ values of about 20 and 300 μs at pH 8 and 23°C. Spectrophotometric experiments show that oxidized ba 3 reacts very poorly with hydrogen peroxide or any of the other exogenous heme iron ligands studied like cyanide, sulfide and azide. At the same time, photoreduction of the enzyme by RuBpy triggers the electrogenic reaction with H2O2 with a second order rate constant of ∼2×103 M−1 s−1. The data indicate that single electron reduction of ba 3 oxidase opens the binuclear center of the enzyme for exogenous ligands. The fractional contribution of the protonic electrogenic phases induced by peroxide in cytochrome ba 3 is much less than in bovine oxidase, pointing to a possibility of a different electrogenic mechanism of the ba 3 oxidase as compared to the oxidases of the aa 3‐type.ba3-type cytochrome c oxidase purified from the thermophilic bacterium Thermus thermophilus has been reconstituted in phospholipid vesicles and laser flash-induced generation of a membrane potential by the enzyme has been studied in a microsecond/ms time scale with Ru(II)-tris-bipyridyl complex (RuBpy) as a photoreductant. Flash-induced single electron reduction of the aerobically oxidized ba3 by RuBpy results in two phases of membrane potential generation by the enzyme with tau values of about 20 and 300 microseconds at pH 8 and 23 degrees C. Spectrophotometric experiments show that oxidized ba3 reacts very poorly with hydrogen peroxide or any of the other exogenous heme iron ligands studied like cyanide, sulfide and azide. At the same time, photoreduction of the enzyme by RuBpy triggers the electrogenic reaction with H2O2 with a second order rate constant of approximately 2 x 10(3) M-1 s-1. The data indicate that single electron reduction of ba3 oxidase opens the binuclear center of the enzyme for exogenous ligands. The fractional contribution of the protonic electrogenic phases induced by peroxide in cytochrome ba3 is much less than in bovine oxidase, pointing to a possibility of a different electrogenic mechanism of the ba3 oxidase as compared to the oxidases of the aa3-type.

Inhibition of membrane-bound cytochrome c oxidase by zinc ions : High-affinity Zn2+-binding site at the P-side of the membrane

In the presence of the uncoupler, external zinc ions inhibit rapidly turnover of cytochrome c oxidase reconstituted in phospholipid vesicles or bound to the membrane of intact mitochondria. The effect is promoted by electron leaks into the oxidase during preincubation with Zn2+. Inhibition of liposome‐bound bovine cytochrome oxidase by external Zn2+ titrates with a K i of 1 ± 0.3 μM. Presumably, the Zn2+‐binding group at the positively charged side is not reactive in the oxidized enzyme, but becomes accessible to the cation in some partially reduced state(s) of the oxidase; reduction of CuB is tentatively proposed to be responsible for the effect.

Peroxidase Activity of Mitochondrial Cytochrome c Oxidase

Mitochondrial cytochrome c oxidase is able to oxidize various aromatic compounds like o-dianisidine, benzidine and its derivatives (diaminobenzidine, etc.), p-phenylenediamine, as well as amidopyrine, melatonin, and some other pharmacologically and physiologically active substances via the peroxidase, but not the oxidase mechanism. Although specific peroxidase activity of cytochrome c oxidase is low compared with classical peroxidases, its activity may be of physiological or pathophysiological significance due to the presence of rather high concentrations of this enzyme in all tissues, as well as specific localization of the enzyme in the mitochondrial membrane favoring accumulation of hydrophobic aromatic substances.

Ferrocyanide-peroxidase activity of cytochrome c oxidase

Redox interaction of mitochondrial cytochrome c oxidase (COX) with ferrocyanide/ferricyanide couple is greatly accelerated by polycations, such as poly-l-lysine [Musatov et al. (1991) Biological Membranes 8, 229-234]. This has allowed us to study ferrocyanide oxidation by COX at very high redox potentials of the ferrocyanide/ferricyanide couple either following spectrophotometrically ferricyanide accumulation or measuring proton uptake associated with water formation in the reaction. At low [ferrocyanide]/[ferricyanide] ratios (Eh values around 500 mV) and ambient oxygen concentration, the ferrocyanide-oxidase activity of COX becomes negligibly small as compared to the reaction rate observed with pure ferrocyanide. Oxidation of ferrocyanide under these conditions, is greatly stimulated by H2O2 or ethylhydroperoxide indicating peroxidatic reaction involved. The ferrocyanide-peroxidase activity of COX is strictly polylysine-dependent and is inhibited by heme a3 ligands such as KCN and NaN3. Apparently the reaction involves normal electron pathway, i.e. electron donation through CuA and oxidation via heme a3. The peroxidase reaction shows a pH-dependence similar to that of the cytochrome c oxidase activity of COX. When COX is preequilibrated with excess H2O2, addition of ferrocyanide shifts the initial steady-state concentrations of the Ferryl-Oxo and Peroxy compounds towards approximately 2:1 ratio of the two intermediates. It is suggested that in the peroxidase cycleferrocyanide donates electrons to both P and F intermediates with a comparable efficiency. Isolation of a partial redox activity of COX opens a possibility to study separately proton translocation coupled to the peroxidase half-reaction of the COX reaction cycle. Copyright 1998

A new spectral intermediate in cyanide binding with the oxidized cytochrome c oxidase

Reaction of cyanide with oxidized cytochrome c oxidase at a low concentration of the ligand and pH > 8 reveals an initial phase, not reported earlier, associated with a small blue shift of the absorption spectrum, which is followed by a conventional red shift of the heme a 3 3+. The initial blue shift resembles the spectral changes induced under the same conditions by low concentrations of azide and it is not observed in the presence of 0.3 mM azide. It is suggested that, similarly to NO, cyanide and HN3 cannot only bind to heme a 3 but to Cu2+ B as well, perturbing the spectrum of a 3+ 3 indirectly. A rapid binding to Cu2+ B could provide the long‐sought intermediate in the cyanide reaction with heme a 3+ 3, the existence of which is implied by the Michaelis‐Menten type kinetics of the latter process.

Proton pumping by cytochrome c oxidase is coupled to peroxidase half of its catalytic cycle

The four‐electron reaction cycle of cytochrome oxidase is comprised of an eu‐oxidase phase in which the enzyme receives the first two electrons and reduces oxygen to bound peroxide and a peroxidase phase in which the peroxy state formed in the eu‐oxidase half of the cycle is reduced by the 3rd and 4th electrons to the ferryl‐oxo state and oxidized form, respectively. Here we show that the ferrocyanide‐peroxidase activity of cytochrome c oxidase incorporated in phospholipid vesicles is coupled to proton pumping. The H+/e − ratio for the ferrocyanide‐peroxidase partial reaction is twice higher than for the overall ferrocyanide‐oxidase activity and is close to 2. These results show that proton pumping by COX is confined to the peroxidase part of the enzyme catalytic cycle (transfer of the 3rd and 4th electron) whereas the eu‐oxidase part (transfer of the first two electrons) may not be proton pumping.

Direct regulation of cytochrome c oxidase by calcium ions.

Cytochrome c oxidase from bovine heart binds Ca2+ reversibly at a specific Cation Binding Site located near the outer face of the mitochondrial membrane. Ca2+ shifts the absorption spectrum of heme a, which allowed previously to determine the kinetics and equilibrium characteristics of the binding. However, no effect of Ca2+ on the functional characteristics of cytochrome oxidase was revealed earlier. Here we report that Ca2+ inhibits cytochrome oxidase activity of isolated bovine heart enzyme by 50–60% with Ki of ∼1 µM, close to Kd of calcium binding with the oxidase determined spectrophotometrically. The inhibition is observed only at low, but physiologically relevant, turnover rates of the enzyme (∼10 s−1 or less). No inhibitory effect of Ca2+ is observed under conventional conditions of cytochrome c oxidase activity assays (turnover number >100 s−1 at pH 8), which may explain why the effect was not noticed earlier. The inhibition is specific for Ca2+ and is reversed by EGTA. Na+ ions that compete with Ca2+ for binding with the Cation Binding Site, do not affect significantly activity of the enzyme but counteract the inhibitory effect of Ca2+. The Ca2+-induced inhibition of cytochrome c oxidase is observed also with the uncoupled mitochondria from several rat tissues. At the same time, calcium ions do not inhibit activity of the homologous bacterial cytochrome oxidases. Possible mechanisms of the inhibition are discussed as well as potential physiological role of Ca2+ binding with cytochrome oxidase. Ca2+- binding at the Cation Binding Site is proposed to inhibit proton-transfer through the exit part of the proton conducting pathway H in the mammalian oxidases.

Effect of Calcium Ions on Electron Transfer between Hemes a and a 3 in Cytochrome c Oxidase

Kinetics of the reduction of the hemes in cytochrome c oxidase in the presence of high concentration of ruthenium(III)hexaammine chloride was examined using a stopped-flow spectrophotometer. Upon mixing of the oxidized enzyme with dithionite and Ru(NH3)63+, three well-resolved phases were observed: heme a reduction reaching completion within a few milliseconds is followed by two slow phases of heme a3 reduction. The difference spectrum of heme a3 reduction in the visible region is characterized by a maximum at ∼612 nm, rather than at 603 nm as was believed earlier. It is shown that in the case of bovine heart cytochrome c oxidase containing a special cation-binding site in which reversible binding of calcium ion occurs, heme a3 reduction is slowed down by low concentrations of Ca2+. The effect is absent in the case of the bacterial cytochrome oxidase in which the cation-binding site contains a tightly bound Ca2+ ion. The data corroborate the inhibition of the cytochrome oxidase enzymatic activity by Ca2+ ions discovered earlier and indicate that the cation affects intramolecular electron transfer.