The operation of the alternative electron-leak pathways mediated by cytochrome c in mitochondria

Low (1 x 10(-9)M) concentrations of cytochrome c inhibit H2O2 production in cytochrome c-depleted mitochondria, purified succinate-cytochrome c reductase (SCR) and antimycin A inhibited cytochrome c-depleted HMP. At higher concentration (2 x 10(-6)M), cytochrome c eliminates pre-existed H2O2 if feeding electrons to it by succinate. Cytochrome c also decreases the OH* produced by succinate-cytochrome c reductase oxidizing succinate. We conclude that the alternative electron-leak pathway mediated by cytochrome c operates very well. In the presence of antimycin A, ferrocytochrome c can suppress the generation of H2O2 in SCR system, but ferricytochrome c cannot. Similar results are obtained on the elimination of pre-existed H2O2 by cytochrome c. For hydroxyl radical, antimycin A abolishes the suppression caused by both ferrocytochrome c and ferricytochrome c. These results indicate that the reductive state of cytochrome c caused by electron-flow is necessary and sufficient for the operation of cytochrome c-mediated electron-leakage pathway.     

Kinetics of interprotein electron transfer between cytochrome c6 and the soluble CuA domain of cyanobacterial cytochrome c oxidase

Cytochrome c6 is a soluble metalloprotein located in the periplasmic space and the thylakoid lumen of many cyanobacteria and is known to carry electrons from cytochrome b6f to photosystem I. The CuA domain of cytochrome c oxidase, the terminal enzyme which catalyzes the four-electron reduction of molecular oxygen in the respiratory chains of mitochondria and many bacteria, also has a periplasmic location. In order to test whether cytochrome c6 could also function as a donor for cytochrome c oxidase, we investigated the kinetics of the electron transfer between recombinant cytochrome c6 (produced in high yield in Escherichia coli by coexpressing the maturation proteins encoded by the ccmA-H gene cluster) and the recombinant soluble CuA domain (i.e., the donor binding and electron entry site) of subunit II of cytochrome c oxidase from Synechocystis PCC 6803. The forward and the reverse electron transfer reactions were studied by the stopped-flow technique and yielded apparent bimolecular rate constants of (3.3 +/- 0.3) x 10(5) M(-1) s(-1) and (3.9 +/- 0.1) x 10(6) M(-1) s(-1), respectively, in 5 mM potassium phosphate buffer, pH 7, containing 20 mM potassium chloride and 25 degrees C. This corresponds to an equilibrium constant Keq of 0.085 in the physiological direction (DeltarG'0 = 6.1 kJ/mol). The reduction of the CuA fragment by cytochrome c6 is almost independent on ionic strength, which is in contrast to the reaction of the CuA domain with horse heart cytochrome c, which decreases with increasing ionic strength. The findings are discussed with respect to the potential role of cytochrome c6 as mobile electron carrier in both cyanobacterial electron transport pathways.     

Depletion of cardiolipin and cytochrome c during ischemia increases hydrogen peroxide production from the electron transport chain

Mitochondrial electron transport is a major source of reactive oxygen species (ROS) during cardiac ischemia and reperfusion. In the isolated rabbit heart, 30 and 45 min of ischemia decrease the contents of cardiolipin and cytochrome c in subsarcolemmal mitochondria (SSM) located beneath the plasma membrane. In contrast, interfibrillar mitochondria (IFM) in the interior of the myocyte do not sustain a decrease in cardiolipin. We proposed that the depletion of cardiolipin and the accompanying cytochrome c loss during ischemia were critical events that amplified ROS production by mitochondria. The total production of H2O2 was measured in submitochondrial particles (SMP) prepared from rabbit heart SSM and IFM after 0, 15, 30, and 45 min of ischemia. With NADH as substrate, total H2O2 production was increased only in SMP from SSM after 30 and 45 min ischemia, when ischemia decreased the content of cardiolipin and cytochrome c. In contrast, ischemia did not augment H2O2 generation in SMP from IFM with preserved cardiolipin and cytochrome c content. Thus, during the evolution of ischemic injury, H2O2 production from the electron transport chain increased after depletion of cardiolipin and the loss of cytochrome c.     

Interaction of cytochrome c with reaction centers of Rhodopseudomonas sphaeroides R-26: localization of the binding site by chemical cross-linking and immunochemical studies

The location of the cytochrome binding site on the reaction center of Rhodopseudomonas sphaeroides was studied by two different approaches. In one, cross-linking agents, principally dithiobis(propionimidate) and dimethyl suberimidate, were used to link cytochrome c and cytochrome c2 to reaction centers; in the other, the inhibition of electron transfer by antibodies against the subunits was investigated. Cytochrome c (horse) cross-linked to the L and M subunits, whereas cytochrome c2 (R. sphaeroides) cross-linked only to the L subunit. The cross-linked reaction center-cytochrome complexes were isolated by affinity chromatography. The rate of electron transfer in the cross-linked cytochrome c2 complex was the same as that in the un-cross-linked complex. However, when cytochrome c was used, the rate in the cross-linked complex was about 15 times slower than that in the un-cross-linked complex. Fab fragments of antibodies specific against the L and M subunits blocked electron transfer from both cytochrome c (horse) and cytochrome c2 (R. sphaeroides). Antibodies specific for the H subunit did not block either reaction. We conclude that the cytochrome binding site on the reaction center is close (approximately 10 A) to both the L and M subunits, possibly in a cleft between them.     

Design of a ruthenium-labeled cytochrome c derivative to study electron transfer with the cytochrome bc1 complex

A new ruthenium-cytochrome c derivative was designed to study electron transfer from cytochrome bc1 to cytochrome c (Cc). The single sulfhydryl on yeast H39C;C102T iso-1-Cc was labeled with Ru(2,2'-bipyrazine)2(4-bromomethyl-4'-methyl-2,2'-bipyridine) to form Ru(z)-39-Cc. The Ru(z)-39-Cc derivative has the same steady-state activity with yeast cytochrome bc1 as wild-type yeast iso-1-Cc, indicating that the ruthenium complex does not interfere in the binding interaction. Laser excitation of reduced Ru(z)-39-Cc results in electron transfer from heme c to the excited state of ruthenium with a rate constant of 1.5 x 10(6) x s(-1). The resulting Ru(I) is rapidly oxidized by atmospheric oxygen in the buffer. The yield of photooxidized heme c is 20% in a single flash. Flash photolysis of a 1:1 complex between reduced yeast cytochrome bc1 and Ru(z)-39-Cc at low ionic strength leads to rapid photooxidation of heme c, followed by intracomplex electron transfer from cytochrome c1 to heme c with a rate constant of 1.4 x 10(4) x s(-1). As the ionic strength is raised above 100 mM, the intracomplex phase disappears, and a new phase appears due to the bimolecular reaction between solution Ru-39-Cc and cytochrome bc1. The interaction of yeast Ru-39-Cc with yeast cytochrome bc1 is stronger than that of horse Ru-39-Cc with bovine cytochrome bc1, suggesting that nonpolar interactions are stronger in the yeast system.     

Topology of beef heart cytochrome c oxidase from studies on reconstituted membranes

The orientation of purified beef heart cytochrome c oxidase, incorporated into vesicles by the cholate dialysis procedure [Carroll, R.C., & Racker, E. (1977) J. Biol. Chem. 252, 6981], has been investigated by functional and structural approaches. The level of heme reduction obtained by using cytochrome c along with the membrane-impermeant electron donor ascorbate was 78 +/- 2% of that obtained with cytochrome c and the membrane-permeant reagent N,N,N',N'-tetramethyl-p-phenylenediamine. Electron transfer from cytochrome c is known to occur exclusively from the outer surface of the mitochondrial inner membrane (C side), implying that at least 78% of the oxidase molecules are oriented in the same way in these vesicles as in the intact mitochondria. Trypsin, which cleaves subunit IV near its N terminus, modifies only 5-7% of this subunit in intact vesicles. This removal of the N-terminal residues has been shown to occur only in mitochondrial membranes with their inner side (M side) exposed. Diazobenzene [35S]sulfonate [( 35S]DABS) likewise modifies subunit IV only in submitochondrial particles. Labeling of intact membranes with [35S]DABS resulted in incorporation of only 4-8% of the total counts that could be incorporated into this subunit in membranes made leaky to the reagent by addition of 2% Triton X-100. Therefore, both the functional and structural data show that at least 80% and probably more of the cytochrome c oxidase molecules are oriented with their C domain outermost and M domains in the lumen of vesicles prepared by the cholate dialysis method.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochrome c-catalyzed oxidation of organic molecules by hydrogen peroxide.

Cytochrome c catalyzed the oxidation of various electron donors in the presence of hydrogen peroxide (H2O2), including 2-2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 4-aminoantipyrine (4-AP), and luminol. With ferrocytochrome c, oxidation reactions were preceded by a lag phase corresponding to the H2O2-mediated oxidation of cytochrome c to the ferric state; no lag phase was observed with ferricytochrome c. However, brief preincubation of ferricytochrome c with H2O2 increased its catalytic activity prior to progressive inactivation and degradation. Superoxide (O2-) and hydroxyl radical (.OH) were not involved in this catalytic activity, since it was not sensitive to superoxide dismutase (SOD) or mannitol. Free iron released from the heme did not play a role in the oxidative reactions as concluded from the lack of effect of diethylenetriaminepentaacetic acid. Uric acid and tryptophan inhibited the oxidation of ABTS, stimulation of luminol chemiluminescence, and inactivation of cytochrome c. Our results are consistent with an initial activation of cytochrome c by H2O2 to a catalytically more active species in which a high oxidation state of an oxo-heme complex mediates the oxidative reactions. The lack of SOD effect on cytochrome c-catalyzed, H2O2-dependent luminol chemiluminescence supports a mechanism of chemiexcitation whereby a luminol endoperoxide is formed by direct reaction of H2O2 with an oxidized luminol molecule, either luminol radical or luminol diazoquinone.     

Role of ubiquinone in the mitochondrial generation of hydrogen peroxide.

Antimycin-inhibited bovine heart submitochondrial particles generate O2- and H2O2 with succinate as electron donor. H2O2 generation involves the action of the mitochondrial superoxide dismutase, in accordance with the McCord & Fridovich [(1969) j. biol. Chem. 244, 6049-6055] reaction mechanism. Removal of ubiquinone by acetone treatment decreases the ability of mitochondrial preparations to generate O2- and H2O2, whereas supplementation of the depleted membranes with ubiquinone enhances the peroxide-generating activity in the reconstituted membranes. Addition of superoxide dismutase to ubiquinone-reconstituted membranes is essential in order to obtain maximal rates of H2O2 generation since the acetone treatment of the membranes apparently inactivates (or removes) the mitochondrial superoxide dismutase. Parallel measurements of H2O2 production, succinate dehydrogenase and succinate-cytochrome c reductase activities show that peroxide generation by ubiquinone-supplemented membranes is a monotonous function of the reducible ubiquinone content, whereas the other two measured activities reach saturation at relatively low concentrations of reducible quinone. Alkaline treatment of submitochondrial particles causes a significant decrease in succinate dehydrogenase activity and succinate-dependent H2O2 production, which contrasts with the increase of peroxide production by the same particles with NADH as electron donor. Solubilized succinate dehydrogenase generates H2O2 at a much lower rate than the parent submitochondrial particles. It is postulated that ubisemiquinone (and ubiquinol) are chiefly responsible for the succinate-dependent peroxide production by the mitochondrial inner membrane.     

Kinetics and mechanism of electron transfer from dithionite to microsomal cytochrome b5 and to forms of the protein associated with charged and neutral vesicles.

The kinetics of the dithionite reduction of calf liver microsomal cytochrome b5, both free in solution and bound to dimyristoyl phosphatidylcholine vesicles, are consistent with electron transfer between SO2- and the exposed haem edge of the protein. The vesicle membrane does not hinder the approach of SO2- to the site of electron transfer on the protein. In 0.01 M-Tris/HCl buffer, pH 8.1, ket (25 degrees C), delta H et and delta S et are estimated to be 1.44 x 10(6) M-1.s-1, 7.8 kJ.mol-1 and -92.3 J.K-1.mol-1 respectively. The cytochrome exhibits an acid dissociation, pKa 9.3 +/- 0.3, and the rate of electron transfer from dithionite to the high-pH form is about one-third of that to the neutral-pH form. The effect of ionic strength on the kinetics is consistent with a reaction between like-charged species and is discussed in terms of a number of theoretical models. In systems comprising cytochrome b5 and negatively charged vesicles, the effect of increasing the charge density of mixed dimyristoyl phosphatidylcholine/dicetyl phosphate vesicles and of increasing the concentration of dicetyl phosphate vesicles is to lower the rate of electron transfer from dithionite to the haem moiety of the cytochrome. With vesicles of high charge density, however, the kinetics are complicated by vesicle-induced conformation changes of the cytochrome.     

Kinetic barriers to the folding of horse cytochrome C in the reduced state

To determine the kinetic barrier in the folding of horse cytochrome c, a CO-liganded derivative of cytochrome c, called carbonmonoxycytochrome c, has been prepared by exploiting the thermodynamic reversibility of ferrocytochrome c unfolding induced by guanidinium hydrochloride (GdnHCl), pH 7. The CO binding properties of unfolded ferrocytochrome c, studied by 13C NMR and optical spectroscopy, are remarkably similar to those of native myoglobin and isolated chains of human hemoglobin. Equilibrium unfolding transitions of ferrocytochrome c in the presence and the absence of CO observed by both excitation energy transfer from the lone tryptophan to the ferrous heme and far-UV circular dichroism (CD) indicate no accumulation of structural intermediates to a detectable level. Values of thermodynamic parameters obtained by two-state analysis of fluorescence transitions are DeltaG(H2O) = 11.65(+/-1.13) kcal x mol(-1) and C(m) = 3.9(+/-0.1) M GdnHCl in the presence of CO, and DeltaG(H2O)=19.3(+/-0.5) kcal x mol(-1) and C(m) = 5.1(+/-0.1) M GdnHCl in the absence of CO, indicating destabilization of ferrocytochrome c by approximately 7.65 kcal x mol(-1) due to CO binding. The native states of ferrocytochrome c and carbonmonoxycytochrome c are nearly identical in terms of structure and conformation except for the Fe2+-M80 --> Fe2+-CO replacement. Folding and unfolding kinetics as a function of GdnHCl, studied by stopped-flow fluorescence, are significantly different for the two proteins. Both refold fast, but carbonmonoxycytochrome c refolds 2-fold faster (tau = 1092 micros at 10 degrees C) than ferrocytochrome c. Linear extrapolation of the folding rates to the ordinate of the chevron plot projects this value of tau to 407 micros. The unfolding rate of the former in water, estimated by extrapolation, is faster by more than 10 orders of magnitude. Significant differences are also observed in rate-denaturant gradients in the chevron. Formation and disruption of the Fe2+-M80 coordination contact clearly impose high-energy kinetic barriers to folding and unfolding of ferrocytochrome c. The unfolding barrier due to the Fe2+-M80 bond provides sufficient kinetic stability to the native state of ferrocytochrome c to perform its physiological function as an electron donor.     

Kinetic studies of the reduction of Pseudomonas aeruginosa ferricytochrome c551 by Fe(EDTA)2-

Kinetic studies of the reduction of Pseudomonas aeruginosa ferricytochrome c551 by Fe(EDTA)2- have been made. The reaction was found to follow a second-order rate law: k 4.2 x 10(3) M(-1) s(-1) [25 degrees, micro0.1 M, pH 7.0 (phosphate)]; deltaH+/+ 3.2 kcal/ mol; AS+/+ -30 cal/mol-deg. The electrostatics-corrected self-exchange rate constant (k11 corr) calculated for cytochrome c551 based on the Fe(EDTA)2- cross reaction is 2 M(-1) s(-1), as compared to a value of 6 M(-1) s(-1) for horse heart cytochrome c. The close correspondence of the two k11 corr values is taken as an indication that the two proteins employ very similar electron transfer mechanisms in their reactions with Fe(EDTA)(2-). It is proposed that this mechanism involves reagent contact, but little protein conformational change, at the partially exposed heme edge.     

Energetics of ATP-driven reverse electron transfer from cytochrome c to fumarate and from succinate to NAD in submitochondrial particles

The maximum Gibbs free energies of reverse electron transfer from succinate to NAD+ and from cytochrome c to fumarate driven by ATP hydrolysis in submitochondrial particles from beef heart were measured as a function of the Gibbs free energy of ATP hydrolysis. The ratio of the energies delta G'redox/delta G'ATP was 1.40 from succinate to NAD+ and 0.89 from cytochrome c to succinate. The ratio, equivalent to a thermodynamic P/2e-ratio, was dependent on whether the electrochemical proton gradient was primarily a membrane potential or a pH gradient for the cytochrome c to fumarate reaction. The results are consistent with H+/ATP = 3 for F1 ATPase, H+/2e- = 4 for NADH-CoQ reductase, and H+(matrix)/2e- = 2 for succinate-cytochrome c reductase.     

Control of electron transfer in the cytochrome system of mitochondria by pH, transmembrane pH gradient and electrical potential. The cytochromes b-c segment.

1. A study is presented of the effects of pH, transmembrane pH gradient and electrical potential on oxidoreductions of b and c cytochromes in ox heart mitochondria and 'inside-out' submitochondrial particles. 2. Kinetic analysis shows that, in mitochondria at neutral pH, there is a restraint on the aerobic oxidation of cytochrome b566 with respect to cytochrome b562. Valinomycin plus K+ accelerates cytochrome b566 oxidation and retards net oxidation of cytochrome b562. At alkaline pH the rate of cytochrome b566 oxidation approaches that of cytochrome b562 and the effects of valinomycin on b cytochromes are impaired. 3. At slightly acidic pH, oxygenation of antimycin-supplemented mitochondria causes rapid reduction of cytochrome b566 and small delayed reduction of cytochrome b562. Valinomycin or a pH increase in the medium promote reduction of cytochrome b562 and decrease net reduction of cytochrome b566. 4. Addition of valinomycin to mitochondria and submitochondrial particles in the respiring steady state causes, at pH values around neutrality, preferential oxidation of cytochrome b566 with respect to cytochrome b562. The differential effect of valinomycin on oxidation of cytochromes b566 and b562 is enhanced by substitution of 1H2O of the medium with 2H2O and tends to disappear as the pH of the medium is raised to alkaline values. 5. Nigericin addition in the aerobic steady state causes, both in mitochondria and submitochondrial particles, preferential oxidation of cytochrome b562 with respect to cytochrome b566. This is accompanied by c cytochrome oxidation in mitochondria but c cytochrome reduction in submitochondrial particles. 6. In mitochondria as well as in submitochondrial particles, the aerobic transmembrane potential (delta psi) does not change by raising the pH of the external medium from neutrality to alkalinity. The transmembrane pH gradient (delta pH) on the other hand, decrease slightly. 7. The results presented provide evidence that the delta psi component of the aerobic delta microH+ (the sum of the proton chemical and electrical activities) exerts a pH-dependent constraint on forward electron flow from cytochrome b566 to cytochrome b562. This effect is explained as a consequence of anisotropic location of cytochromes b566 and b562 in the membrane and the pH-dependence of the redox function of these cytochromes. Transmembrane delta pH, on the other hand, exerts control on electron flow from cytochrome b562 to c cytochromes.     

Production of reactive oxygen species by mitochondria: central role of complex III

The mitochondrial respiratory chain is a major source of reactive oxygen species (ROS) under pathological conditions including myocardial ischemia and reperfusion. Limitation of electron transport by the inhibitor rotenone immediately before ischemia decreases the production of ROS in cardiac myocytes and reduces damage to mitochondria. We asked if ROS generation by intact mitochondria during the oxidation of complex I substrates (glutamate, pyruvate/malate) occurred from complex I or III. ROS production by mitochondria of Sprague-Dawley rat hearts and corresponding submitochondrial particles was studied. ROS were measured as H2O2 using the amplex red assay. In mitochondria oxidizing complex I substrates, rotenone inhibition did not increase H2O2. Oxidation of complex I or II substrates in the presence of antimycin A markedly increased H2O2. Rotenone prevented antimycin A-induced H2O2 production in mitochondria with complex I substrates but not with complex II substrates. Catalase scavenged H2O2. In contrast to intact mitochondria, blockade of complex I with rotenone markedly increased H2O2 production from submitochondrial particles oxidizing the complex I substrate NADH. ROS are produced from complex I by the NADH dehydrogenase located in the matrix side of the inner membrane and are dissipated in mitochondria by matrix antioxidant defense. However, in submitochondrial particles devoid of antioxidant defense ROS from complex I are available for detection. In mitochondria, complex III is the principal site for ROS generation during the oxidation of complex I substrates, and rotenone protects by limiting electron flow into complex III.     

Topology of subunits of the mammalian cytochrome c oxidase: relationship to the assembly of the enzyme complex.

The arrangement of three subunits of beef heart cytochrome c oxidase, subunits Va, VIa, and VIII, has been explored by chemical labeling and protease digestion studies. Subunit Va is an extrinsic protein located on the C side of the mitochondrial inner membrane. This subunit was found to label with N-(4-azido-2-nitrophenyl)-2-aminoethane[35S]sulfonate and sodium methyl 4-[3H]formylphenyl phosphate in reconstituted vesicles in which 90% of cytochrome c oxidase complexes were oriented with the C domain outermost. Subunit VIa was cleaved by trypsin both in these reconstituted vesicles and in submitochondrial particles, indicating a transmembrane orientation. The epitope for a monoclonal antibody (mAb) to subunit VIa was lost or destroyed when cleavage occurred in reconstituted vesicles. This epitope was localized to the C-terminal part of the subunit by antibody binding to a fusion protein consisting of glutathione S-transferase (G-ST) and the C-terminal amino acids 55-85 of subunit VIa. No antibody binding was obtained with a fusion protein containing G-ST and the N-terminal amino acids 1-55. The mAb reaction orients subunit VIa with its C-terminus in the C domain. Subunit VIII was cleaved by trypsin in submitochondrial particles but not in reconstituted vesicles. N-Terminal sequencing of the subunit VIII cleavage product from submitochondrial particles gave the same sequence as the untreated subunit, i.e., ITA, indicating that it is the C-terminus which is cleaved from the M side. Subunits Va and VIII each contain N-terminal extensions or leader sequences in the precursor polypeptides; subunit VIa is made without an N-terminal extension.     

The modulation of cytochrome c electron self-exchange by site-specific chemical modification and anion binding

The site-specific chemical modification of horse heart cytochrome c at Lys-13 and -72 using 4-chloro-3,5-dinitrobenzoic acid (CDNB) increases the electron self-exchange rate of the protein. In the presence of 0.24 M cacodylate (pH* 7.0) the electron self-exchange rate constants, kex, measured by a 1H NMR saturation transfer method at 300 K, are 600, 6 X 10(3) and 6 X 10(4) M-1 X s-1 for native, CDNP-K13 and CDNP-K72 cytochromes c respectively. Repulsive electrostatic interactions, which inhibit cytochrome c electron self-exchange, are differentially affected by modification. Measurements of 1H NMR line broadening observed with partially oxidised samples of native cytochrome c show that ATP and the redox inert multivalent anion Co(CN)3-6 catalyse electron self-exchange. At saturation a limiting value of approximately 1.4 X 10(5) M-1 X s-1 is observed for both anions.     

Thiosulfate Oxidation and Electron Transport in Thiobacillus novellus.

Aleem, M. I. H. (Research Institute for Advanced Studies, Baltimore, Md.). Thiosulfate oxidation and electron transport in Thiobacillus novellus. J. Bacteriol. 90:95-101. 1965.-A cell-free soluble enzyme system capable of oxidizing thiosulfate was obtained from Thiobacillus novellus adapted to grow autotrophically. The enzyme systems of autotrophically grown cells brought about the transfer of electrons from thiosulfate to molecular oxygen via cytochromes of the c and a types; the reactions were catalyzed jointly by thiosulfate oxidase and thiosulfate cytochrome c reductase. The levels of both of these enzymes were markedly reduced in the heterotrophically grown organism. Cell-free extracts from the autotrophically grown T. novellus catalyzed formate oxidation and enzymatically reduced cytochrome c with formate. Both formate oxidation and cytochrome c reduction activities were abolished under heterotrophic conditions. The thiosulfate-activating enzyme S(2)O(3) (-2)-cytochrome c reductase, as well as thiosulfate oxidase, was localized chiefly in the soluble cell-free fractions, and the former enzyme was purified more than 200-fold by ammonium sulfate fractionation and calcium phosphate gel adsorption procedures. Optimal activity of the purified enzyme occurred at pH 8.0 in the presence of 1.67 x 10(-1)m S(2)O(3) (-2) and 2.5 x 10(-4)m cytochrome c. The thiosulfate oxidase operated optimally at pH 7.5 and thiosulfate concentrations of 1.33 x 10(-3) to 3.33 x 10(-2)m in the presence of added cytochrome c at a concentration of 5 x 10(-4)m. Both enzymes were markedly sensitive to cyanide and to a lesser extent to some metal-binding agents. Although a 10(-3)m concentration of p-hydroxymercuribenzoate had no effect on S(2)O(3) (-2)-cytochrome c reductase, it caused a 50% inhibition of S(2)O(3) (-2) oxidase, which was completely reversed in the presence of 10(-3)m reduced glutathione. Carbon monoxide also inhibited S(2)O(3) (-2) oxidase; the inhibition was completely reversed by light.     

Copper,zinc superoxide dismutase as a univalent NO(-) oxidoreductase and as a dichlorofluorescin peroxidase.

Nitroxyl (NO(-)) may be produced by nitric-oxide synthase and by the reduction of NO by reduced Cu,Zn-SOD. The ability of NO(-) to cause oxidations and of SOD to inhibit such oxidations was therefore explored. The decomposition of Angeli's salt (AS) produces NO(-) and that in turn caused the aerobic oxidation of NADPH, directly or indirectly. O(2) was produced concomitant with the aerobic oxidation of NADPH by AS, as evidenced by the SOD-inhibitable reduction of cytochrome c. Both Cu,Zn-SOD and Mn-SOD inhibited the aerobic oxidation of NADPH by AS, but the amounts required were approximately 100-fold greater than those needed to inhibit the reduction of cytochrome c. This inhibition was not due to a nonspecific protein effect or to an effect of those large amounts of the SODs on the rate of decomposition of AS. NO(-) caused the reduction of the Cu(II) of Cu,Zn-SOD, and in the presence of O(2), SOD could catalyze the oxidation of NO(-) to NO. The reverse reaction, i.e. the reduction of NO to NO(-) by Cu(I),Zn-SOD, followed by the reaction of NO(-) with O(2) would yield ONOO(-) and that could explain the oxidation of dichlorofluorescin (DCF) by Cu(I),Zn-SOD plus NO. Cu,Zn-SOD plus H(2)O(2) caused the HCO(3)(-)-dependent oxidation of DCF, casting doubt on the validity of using DCF oxidation as a reliable measure of intracellular H(2)O(2) production.     

Respiratory electron transfer activity in an asolectin-isooctane reverse micellar system.

Bovine heart submitochondrial particles (SMP) were solubilized in an asolectin isooctane reverse micellar system and the functionality of the respiratory chain was tested by spectroscopic and amperometric techniques. Electron transfer rate supported by NADH was very slow as evidenced by the low cytochrome reduction levels attained over long incubation periods. In the presence of KCN, NADH caused 34% and 12.5% reduction of the cytochromes aa3 and c, respectively, and negligible reduction of cytochrome b. Supplementation of the system with menadione rose the NADH-dependent reduction of all the cytochromes to levels that were close to the total content. However, no measurable O2 uptake activity took place in the presence of NADH plus menadione, or with ascorbate (or NADH) plus TMPD reducing systems. Therefore, it is suggested that in the organic medium, electron transfer from NADH to O2 is arrested at the terminal oxidase step. Cytochrome oxidase reduced by ascorbate (or NADH) plus TMPD seems to be trapped in its half reduced state (ie, a2+ a3(3+)). Although it is poorly reactive with O2, it can transfer electrons back to cytochrome c and TMPD. The electron transfer block to O2 was overcome when PMS was used instead of TMPD. This seems to be due to the recognized capacity of PMSH2 to carry out simultaneous reduction of both a CuA and a3 CuB redox centers of cytochrome oxidase. The cytochrome oxidase reaction in the organic solvent was highly sensitive to KCN (Ki 1.9 microM) and showed bell-shaped kinetics towards the PMS concentration and a sigmoidal response to water concentration, reaching its maximal turnover number (18 s-1) at 4 mM PMS and 1.1% (v/v) water.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutants of the CuA site in cytochrome c oxidase of Rhodobacter sphaeroides: II. Rapid kinetic analysis of electron transfer

The function of the binuclear Cu(A) center in cytochrome c oxidase (CcO) was studied using two Rhodobacter sphaeroides CcO mutants involving direct ligands of the Cu(A) center, H260N and M263L. The rapid electron-transfer kinetics of the mutants were studied by flash photolysis of a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine-55. The rate constant for intracomplex electron transfer from heme c to Cu(A) was decreased from 40000 s(-1) for wild-type CcO to 16000 s(-1) and 11000 s(-1) for the M263L and H260N mutants, respectively. The rate constant for electron transfer from Cu(A) to heme a was decreased from 90000 s(-1) for wild-type CcO to 4000 s(-1) for the M263L mutant and only 45 s(-1) for the H260N mutant. The rate constant for the reverse reaction, heme a to Cu(A), was calculated to be 66000 s(-1) for M263L and 180 s(-1) for H260N, compared to 17000 s(-1) for wild-type CcO. It was estimated that the redox potential of Cu(A) was increased by 120 mV for the M263L mutant and 90 mV for the H260N mutant, relative to the potential of heme a. Neither mutation significantly affected the binding interaction with cytochrome c. These results indicate that His-260, but not Met-263, plays a significant role in electron transfer between Cu(A) and heme a.