Natalia V. Azarkina

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).

A cytochrome bb'-type quinol oxidase in Bacillus subtilis strain 168

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa 3, which is a cytochromec oxidase, and cytochrome aa 3 andbd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa 3 andcaa 3 terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bdcontent of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a “cytochrome o”-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb′ type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB andythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion ofythAB in strain LUH27 or the presence of theyth genes on plasmid did not affect the expression of thebb′ oxidase. It is concluded that the novelbb′-type oxidase probably is cytochrome bdencoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e.cytochromeb 558 b 595 b′.

A bb´-type quinol oxidase in Bacillus subtilis strain 168

The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa 3, which is a cytochromec oxidase, and cytochrome aa 3 andbd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa 3 andcaa 3 terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bdcontent of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a “cytochrome o”-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb′ type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB andythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion ofythAB in strain LUH27 or the presence of theyth genes on plasmid did not affect the expression of thebb′ oxidase. It is concluded that the novelbb′-type oxidase probably is cytochrome bdencoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e.cytochromeb 558 b 595 b′.

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.

Energization of Bacillus subtilis membrane vesicles increases catalytic activity of succinate: Menaquinone oxidoreductase

In this work, high ΔμH+-dependent succinate oxidase activity has been demonstrated for the first time with membrane vesicles isolated from Bacillus subtilis. The maximal specific rate of succinate oxidation by coupled inside-out membrane vesicles isolated from a B. subtilis strain overproducing succinate:menaquinone oxidoreductase approaches the specific rate observed with the intact cells. Deenergization of the membrane vesicles with ionophores or alamethicin brings about an almost complete inhibition of succinate oxidation. An apparent Km for succinate during the energy-dependent succinate oxidase activity of the vesicles (2.2 mM) is higher by an order of magnitude than the Km value measured for the energy-independent reduction of 2,6-dichlorophenol indophenol. The data reveal critical importance of ΔμH+ for maintaining active electron transfer by succinate:menaquinone oxidoreductase. The role of ΔμH+ might consist in providing energy for thermodynamically unfavorable menaquinone reduction by succinate by virtue of transmembrane electron transport within the enzyme down the electric field; alternatively, ΔμH+ could play a regulatory role by maintaining the electroneutrally operating enzyme in a catalytically active conformation.

Peculiarities of cyanide binding to the ba3-type cytochrome oxidase from the thermophilic bacterium Thermus thermophilus

Cytochrome c oxidase of the ba3-type from Thermus thermophilus does not interact with cyanide in the oxidized state and acquires the ability to bind heme iron ligands only upon reduction. Cyanide complexes of the reduced heme a3 in cytochrome ba3 and in mitochondrial aa3-type cytochrome oxidase are similar spectroscopically, but the a32+-CN complex of cytochrome ba3 is strikingly tight. Experiments have shown that the Kd value of the cytochrome ba3 complex with cyanide in the presence of reductants of the enzyme binuclear center does not exceed 10−8 M, which is four to five orders of magnitude less than the Kd of the cyanide complex of the reduced heme a3 of mitochondrial cytochrome oxidase. The tightness of the cytochrome ba3 complex with cyanide is mainly associated with an extremely slow rate of the ligand dissociation (koff ≤ 10−7 sec−1), while the rate of binding (kon ∼ 102 M−1·sec−1) is similar to the rate observed for the mitochondrial cytochrome oxidase. It is proposed that cyanide dissociation from the cytochrome ba3 binuclear center might be hindered sterically by the presence of the second ligand molecule in the coordination sphere of CuB2+. The rate of cyanide binding with the reduced heme a3 does not depend on pH in the neutral area, but it approaches linear dependence on H+ activity in the alkaline region. Cyanide binding appears to be controlled by protonation of an enzyme group with pKa = 8.75.

Calcium ions inhibit reduction of heme a in bovine cytochrome c oxidase

The effect of Ca2+ on the rate of heme a reduction by dithionite and hexaammineruthenium (RuAm) was studied in the cyanide‐complexed bovine cytochrome oxidase (CcO). The rate of heme a reduction is proportional to RuAm concentration below 300 μM with k v of 0.53 × 106 M−1s−1. Ca2+ inhibits the rate of heme a reduction by dithionite by ∼25%. As the reaction speeds up with increased concentrations of RuAm, the inhibition by Ca2+ disappears. The inhibition of heme a reduction may contribute to recently described partial inhibition of CcO by Ca2+ in the enzymatic assays. The inhibitory effect of Ca2+ on heme a reduction indicates that ET through heme a may be coupled to proton movement in the exit part of the proton channel H.