Alexander A. Konstantinov

Effect of electron transfer inhibitors on superoxide generation in the cytochrome bc1 site of the mitochondrial respiratory chain.

Antimycin, 2‐nonyl‐4‐hydroxyquinoline N‐oxide and funiculosin induce O·− 2 generation by submitochondrial particles oxidizing succinate, whereas KCN, mucidin, myxothiazol or 2,3‐dimercaptopropanol inhibit O·− 2 generation. Thenoyltrifluoroacetone does not induce superoxide production by itself but slightly stimulates the reaction initiated by antimycin. The results indicate that auto‐oxidation of unstable ubisemiquinone formed in centre o of the Q‐cycle generates most of the O·− 2 radicals in the cytochrome bc 1‐site of the mitochondrial respiratory chain.

Involvement of intramitochondrial protons in redox reactions of cytochrome a

The energy-conserving function of mitochondrial cytochrome oxidase has been widely believed to consist in the transmembrane transfer of an electron [l-4], as originally proposed by Mitchell [5]. On the other hand, involvement of protons in the cytochrome oxidase reaction was largely neglected (but see [6-8]), although reduction of oxygen to water needs not only 4 ebut also 4 H+ions. We confirmed the important observation [9,10] that Em of the high-potential heme of cytochrome oxidase was pH dependent [ 1 I]. We also found that it was pH inside mitochondria, that was ‘felt’ by I?,,, of cytochrome oxidase [ 111. Since the high-potential component of cytochrome oxidase was thought at that time to represent heme a3 [2,12-141, we interpreted our results as evidence for heme a3 being localized at the inner face of the mitochondrial membrane [ 111. Recent revision of the cytochrome oxidase spectral and potentiometric characteristics [15-171 made us re-investigate the problem. The data reported here (see also [ 181) indicate that: (1) Partial redox-linked protonation is characteristic of both cytochromes a and a3 in the high-potential redox transition of cytochrome oxidase. (2) Cytochrome a, known to react with cytochrome

Superoxide dismutase and glutathione peroxidase activities in tumors

1. Introduction Superoxide dismutase (SOD) is believed to play a key role in the enzymatic defence of the cell against oxygen toxicity, the belief being substantiated by the ubiquitous presence of this enzyme in aerobic orga- nisms and by an impressive body of evidence for a protective role of SOD both in vitro and in vivo (cf. reviews [1,2] ). It was of obvious interest to study SOD activity in tumors. Dionisi et al. [3] reported SOD to be absent from tumor mitochondria. On the other hand, our preliminary data [4] and recent results of Rotiho and collaborators [S] pointed to a low SOD activity in the cytosol of tumor cells. In this communication we confirm and extend our preliminary observations showing tumors to be poor in cytosolic cyanide- sensitive SOD, compared to normal tissues. In addition, a decline in the SOD activity of the Hepatoma 27 mitochondria (as compared to liver mitochondria) is found to be due solely to a lower content of the cytoplasmic-type Cu-Zn-enzyme localized in the intermembrane space of mitochondria; no change in the eumitochondrial cyanide-insensitive enzymatic SOD activity could be observed. The activity of glutathione peroxidase (GP), another enzyme thought to be ‘the first line of defence agsinst oxidative damage’ [6-81,

Superoxide generation by the respiratory chain of tumor mitochondria.

O2-. generation by the succinate oxidase segment of the respiratory chain of mitochondria and submitochondrial particles from hepatoma 22a and hepatoma Zajdela has been studied with the use of the Tiron method. In the presence of succinate, superoxide generation is induced by antimycin, 2-n-4-hydroxyquinoline N-oxide or funiculosin, and is inhibited by mucidin, myxothiazol or cyanide. The rate of O2-. generation in the antimycin-inhibited state is maximal at the [succinate]/[fumarate] ratio of 1:10 and diminishes at more positive and more negative redox potentials. These characteristics of O2-.-generation are the same as observed earlier in submitochondrial particles from normal tissues. Accordingly, the mechanism of superoxide production is suggested to be the same in tumor and normal mitochondria, namely, autoxidation of the unstable ubisemiquinone in the ubiquinol-oxidizing centre o of cytochrome bc1 complex. With respect to the rate of O2-. generation, the hepatoma mitochondrial membranes are approximately twice as active as bovine heart submitochondrial particles and exceed those from rat liver by more than one order of magnitude.

Semiquinone Q in the respiratory chain of electron transport particles: electron spin resonance studies.

The specific role of coenzyme Q semiquinone (QH.) in the respiratory chain has been discussed extensively [l-7] , but few experimental data are available on this point. Though evidence for the QH. contribution to the ESR signal at g 2.00 in mitochondria [8] , ETP [9] and bacteria [lo] had been previously obtained, (for references, see [g-12]) significant interference from flavosemiquinones obscured the results of those early observations. We have recently chosen ETP poised with the succinate/fumarate redox couple as a convenient experimental system for the QH’ ESR studies [ 11 ,121. The rationale for this choice was that at low succinate/ fumarate ratios (Eh +50 mV) reduction of the SDH flavin (Em = -40 mV [13] or -90 mV [14]) and, the more so, of the NADH-dehydrogenase flavin should be thermodynamically unfavourable while the yield of QH* should be about maximal (I!?, QH2/Q = +40 mV at pH 7.4 [15]). Some data

Interaction of the bacterial terminal oxidase cytochrome bd with nitric oxide

Cytochrome bd is a prokaryotic terminal oxidase catalyzing O2 reduction to H2O. The oxygen‐reducing site has been proposed to contain two hemes, d and b 595, the latter presumably replacing functionally CuB of heme‐copper oxidases. We show that NO, in competition with O2, rapidly and potently (K i=100±34 nM at ∼70 μM O2) inhibits cytochrome bd isolated from Escherichia coli and Azotobacter vinelandii in turnover, inhibition being quickly and fully reverted upon NO depletion. Under anaerobic reducing conditions, neither of the two enzymes reveals NO reductase activity, which is proposed to be associated with CuB in heme‐copper oxidases.

H2O2‐Induced Conversion of Cytochrome c Oxidase Peroxy Complex to Oxoferryl State

Addition of high H2O2 concentrations to a peroxy complex of proteoliposome-bound cytochrome oxidase converts the complex to a spectrally distinct species. The difference spectrum of the high-peroxide compound versus the oxidized enzyme is characterized in a visible range by a broad symmetrical band at 580 nm (delta epsilon approximately equal to 4 mM-1 cm-1) with a minor second maximum at approximately 535 nm; a complete disappearance of the 605-607-nm peak occurs which is typical of the peroxy complex. In the Soret band, the spectrum of the high H2O2 compound is virtually indistinguishable from that of the initial peroxide adduct. The high-peroxide compound appears to be identical with an oxoferryl intermediate formed in the forward and reversed cytochrome oxidase reaction. The transition of the peroxy complex to the oxoferryl state is favored by alkaline pH and counteracted by ferricyanide. The peroxy and oxoferryl complexes of cytochrome c oxidase can also be formed with t-butylhydroperoxide.

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).

Electrogenic reduction of the secondary quinone acceptor in chromatophores of Rhodospirillum rubrum: Rapid kinetics measurements

Electron transfer QA → QB has been reconstituted with added Q‐10 in Rhodospirillum rubrum chromatophores associated with a phospholipid‐impregnated collodion film. Rapid kinetics measurements of laser flash‐induced ΔΨ generated in the chromatophores show that whereas electron transfer from Qa − to QB upon the first flash is not electrogenic in dark‐adapted chromatophores, reduction of Q− B to Qbh2 induced by the second flash gives rise to an electrogenic phase with τ = 250 μs at pH 7.5 which contributes about 10% to the total ΔΨ generated upon the flash. The electrogenic phase is ascribed to vectorial protonation of Q2− B.

Redox-linked protolytic reactions in soluble cytochrome-c oxidase from beef-heart mitochondria: redox Bohr effects

A study is presented of co-operative redox-linked protolytic reactions (redox Bohr effects) in soluble cytochrome-c oxidase purified from bovine-heart mitochondria. Bohr effects were analyzed by direct measurement, with accurate spectrophotometric and potentiometric methods, of H+ uptake and release by the oxidase associated with reduction and oxidation of hemes a and a3. CuA and CuB in the unliganded and in the CN- or CO-liganded enzyme. The results show that there are in the bovine oxidase four protolytic groups undergoing reversible pK shifts upon oxido-reduction of the electron transfer metals. Two groups with pKox and pKred values around 7 and > 12 respectively appear to be linked to redox transitions of heme a3. One group with pKox and pKred around 6 and 7 is apparently linked to CuB, a fourth one with pKox and pKred of 6 and 9 appears to be linked to heme a. The possible nature of the amino acids involved in the redox Bohr effects and their role in H+ translocation is discussed.