On the mechanism of production of superoxide radical by reaction mixtures containing NADH, phenazine methosulfate, and nitroblue tetrazolium

In aerobic reaction mixtures containing NADH, phenazine methosulfate, and nitroblue tetrazolium, O2- production is mediated by the tetrazolium, not the phenazine. Thus, superoxide dismutase inhibited reduction of the tetrazolium, but when ferricytochrome c was substituted for the tetrazolium its reduction was not affected by this enzyme. Furthermore, NADH plus the phenazine did not accelerate the oxidation of epinephrine to adrenochrome unless the tetrazolium was present, and under those circumstances superoxide dismutase did inhibit adrenochrome formation. When the tetrazolium and ferricytochrome c were present simultaneously, addition of superoxide dismutase was seen to accelerate the reduction of the cytochrome. This is explainable by the reduction of O2- by the reduced phenazine, which thus competes with cytochrome c for the available O2-. When the O2- was eliminated by superoxide dismutase, more of the reduced phenazine was available for the direct reduction of cytochrome c.     

Aerobic and anaerobic reduction of nitrated pyrenes in vitro

Nitrated pyrenes are mutagenic and tumorigenic environmental pollutants that are activated to DNA-binding derivatives via nitroreduction. We have investigated the enzymatic nitroreduction of 1-nitropyrene, 1,3-, 1,6- and 1,8-dinitropyrene to determine if differences in the extent of nitroreduction may help explain differences in their biological potencies. Each nitrated pyrene was incubated aerobically and anaerobically with 105,000 X g supernatant (S105) from Salmonella typhimurium TA98 and the nitroreductase-deficient strain, TA98NR, and with cytosol and microsomes from rat and human liver. Under anaerobic conditions, 1-nitropyrene and 1,3-dinitropyrene were reduced by TA98 S105 to a lesser extent than 1,6- and 1,8-dinitropyrene. The extent of 1,6- and 1,8-dinitropyrene metabolism was not altered relative to TA98 when using TA98NR S105, but the nitroreduction of 1-nitropyrene and 1,3-dinitropyrene was decreased. Both rat and human liver cytosol and microsomes reduced 1,6- and 1,8-dinitropyrene to greater extents than 1-nitropyrene and 1,3-dinitropyrene. Under aerobic conditions rat and human liver cytosols were similar to TA98 S105 in that aminopyrene decreased while nitrosopyrene formation increased. By comparison, oxygen decreased the microsomal formation of both nitrosopyrenes and aminopyrenes. The reduction of succinoylated cytochrome c was measured during the hepatic metabolism of nitro- and nitrosopyrenes under aerobic conditions. The data indicated that reduced nitro- and nitrosopyrene intermediates were directly reducing succinoylated cytochrome c and that the assay could be used as a measure of aerobic nitroreduction. These studies demonstrate that 1,6- and 1,8-dinitropyrene are reduced to a greater extent than 1-nitropyrene and 1,3-dinitropyrene, which corresponds to their relative biological potencies as mutagens and carcinogens. Furthermore, although more extensive nitroreduction is detected under anaerobic conditions, the nitroreduction that occurs aerobically may be important for the mutagenic and tumorigenic properties of these compounds.     

Redox state-dependent aggregation of mitochondria induced by cytochrome c

Cytochrome c is known to play central role in apoptosis. Here, it is shown that ferricytochrome c, but not ferrocytochrome c is able to directly induce the aggregation of rat liver mitochondria, similar to the effect caused by magnesium ions at high concentrations. The aggregation was revealed by a decrease in light dispersion of mitochondrial suspension and it was confirmed by the optical microscopy. In the medium containing NADH and cytochrome c, mitochondrial aggregation was initiated only after exhaustion of NADH leading to oxidation of cytochrome c. The aggregation induced by 30 μM ferricytochrome c, but not by 5 mM MgCl(2), was completely inhibited by 30-100 μM ferricyanide, thus indicating that ferricyanide-cytochrome c specific interaction prevents mitochondrial aggregation. After completion of the aggregation caused by ferricytochrome c, this effect cannot be readily reversed by subsequent reduction of cytochrome c. The aggregation induced by ferricytochrome c and/or magnesium ions explains masking of the external NADH-oxidase activity of mitochondria in vitro reported in the literature. This new cytochrome c redox state-dependent phenomenon might also be involved in more complex mechanisms controlling aggregation (clustering) of mitochondria in vivo under the influence of pro-apoptotic factors and requires further study.     

A spectrophotometric assay for superoxide dismutase activities in crude tissue fractions.

A sensitive and reliable assay method was developed to characterize crude cell homogenates and subcellular fractions with regard to their superoxide dismutase (SOD) activities. The determination of SOD activities was based on the well-known spectrophotometric assay introduced by McCord & Fridovich [(1969) J. Biol. Chem. 244, 6049-6055], with partially succinylated (3-carboxypropionylated) rather than native ferricytochrome c as indicating scavenger. Partial succinylation of cytochrome c resulted in minimization of interference associated with the interaction of cytochrome c with mitochondrial cytochrome c oxidase or cytochrome c reductases. The further increase in specificity, with regard to exclusion of cytochrome c oxidase interference, gained as a consequence of the high pH of 10 enabled the analysis of samples as rich in cytochrome c oxidase activity as the mitochondrial fraction in the presence or absence of membrane-disrupting detergents. Linear relationships for the dependence of the SOD activities with protein concentration were obtained with rat liver homogenate, mitochondrial and microsomal fractions, indicating negligible interference. Furthermore, by choosing a high pH for the assay medium, a 4-fold increase in sensitivity compared with the classical SOD assay, carried out at pH 7.8, was gained as well as a more precise resolution of Cu/Zn-SOD and Mn-SOD by 2 mM-KCN in samples with a high ratio of Mn-SOD to Cu/Zn-SOD, such as mitochondria. The complete trapping of the O2.- radicals, which was more feasible at pH 10 than at pH 7.8, enabled the application of a simple equation derived for the calculation of appropriately defined units of SOD activity from a single experiment.     

The mechanism of reduction of cytochrome c as studied by pulse radiolysis.

1. The reaction of hydrated electrons with ferricytochrome c was studied using the pulse-radiolysis technique. 2. In 3.3 mM phosphate-buffer (pH 7.2), 100 mM methanol and at a concentration of cytochrome c of less than 20 muM the reduction kinetics of ferricytochrome c by hydrated electrons is a bimolecular process with a rate constant of 4.5-10-10 M-1-S-1 (21 degrees C). 3. At a concentration of cytochrome c of more than 20 muM the apparent order of the reaction of hydrated electrons with ferricytochrome c measured at 650 nm decreases due to the occurrence of a rate-determining first-order process with an estimated rate constant of 5-10-6s-1 (pH 7.2, 21 degrees C). 4. At high concentration of cytochrome c the reaction-time courses measured at 580 and 695 nm appear to be biphasic. A rapid initial phase (75% and 30% of total absorbance change at 580 and 695 nm, respectively), corresponding to the reduction reaction, is followed by a first-order change in absorbance with a rate constant of 1.3-10-5 S-1 (pH 7.2, 21 degrees C). 5. The results are interpreted in a scheme in which first a transient complex between cytochrome c and the hydrated electron is formed, after which the heme iron is reduced and followed by relaxation of the protein from its oxidized to its reduced conformation. 6. It is calculated that one of each three encounters of the hydrated electron and ferricytochrome c results in a reduction of the heme iron. This high reaction probability is discussed in terms of charge and solvent interactions. 7. A reduction mechanism for cytochrome c is favored in which the reduction equivalent from the hydrated electron is transmitted through a specific pathway from the surface of the molecule to the heme iron.     

Reduction of horse heart ferricytochrome c by bovine liver ferrocytochrome b5. Experimental and theoretical analysis.

The reduction of horse heart ferricytochrome c by the tryptic fragment of bovine liver cytochrome b5 and its dimethyl ester heme (DME)-substituted derivative has been studied as a function of ionic strength, pH, and temperature under solution conditions where the reaction is bimolecular. The rate constant for ferricytochrome c reduction by native ferrocytochrome b5 is 1.8 (+/- 0.2) x 10(7) M-1 s-1 (25 degrees C) with delta H++ = 7.5 (+/- 0.2) kcal/mol and delta S++ = -0.3 (+/- 0.6) eu (pH 7.0, I = 0.348 M). Under the same solution conditions, the reduction of ferricytochrome c by DME-ferrocytochrome b5 proceeds with a rate constant of 1.7 (+/- 0.1) x 10(7) M-1 s-1 with delta H++ = 7.9 (+/- 0.4) kcal/mol and delta S++ = 1 (+/- 1) eu. The rate constants for both reactions are strongly dependent on ionic strength. A detailed electrostatic analysis of the proteins has been performed. Two relatively simple Brownian dynamics simulation models predict rate constants for the reaction between the two native proteins that demonstrate a dependence on ionic strength similar to that observed experimentally. In one of these models, the proteins are treated as spheres with reactive surface patches that are defined by a 5 degrees cone generated about the dipole vector calculated for each protein and aligned with the presumed electron-transfer site near the partially exposed heme edge. The second model replaces the reactive patch assumption with an exponential distance dependence for the probability of reaction that permits estimation of a value for the distance-dependence factor alpha. Calculations with this latter model in combination with the aligned dipole assumption provide a reasonable approximation to the observed ionic strength dependence for the reaction and are consistent with a value of alpha = 1.2 A-1.     

Effect of interferon inducers on superoxide anion generation from rat liver microsomes detected by lucigenin chemiluminescence

Microsomal superoxide anion (O2-) production was detected using the chemiluminigenic probe, bis-N-Methylacridinium nitrate (lucigenin). Superoxide dismutase (SOD) inhibited 55% of the light emission but in the presence of a detergent (Triton X100) SOD inhibited the light emission by 94%. Lucigenin chemiluminescence from rat liver microsomes supplemented with NADPH was found to be selective and sensitive in detecting the O2- production. Treatment of rats with poly IC and LPS resulted in a decrease of the hepatic microsomal cytochrome P450 content by 44% and 37% respectively. The decrease in the cytochrome P450 contents was accompanied by a decrease in LgCl from the hepatic microsomal fractions by 61% for the poly IC and by 51% for the LPS treated rats. This is the first report to demonstrate that decreased P450 in the presence of normal amounts of cytochrome P450(c) reductase produce correspondingly less O2- from the microsomes.     

Reactions of the ferri-ferrocytochrome-c system with superoxide/oxygen and CO2-/CO2 studied by fast pulse radiolysis.

The reduction of ferricytochrome c by O2- and CO2- was studied in the pH range 6.6-9.2 and Arrhenius as well as Eyring parameters were derived from the rate constants and their temperature dependence. Ionic effects on the rate indicate that the redox process proceeds through a multiply-positively charged interaction site on cytochrome c. It is shown that the reaction with O2- (and correspondingly with O2 of ferrocytochrome c) is by a factor of approx. 10(3) slower than warranted by factors such as redox potential. Evidence is adduced to support the view that this slowness is connected with the role of water in the interaction between O2-/O2 and ferri-ferrocytochrome c in the positively charged interaction site on cytochrome c in which water molecules are specifically involved in maintaining the local structure of cytochrome c and participate in the process of electron equivalent transfer.     

Superoxide generation by NADPH-cytochrome P-450 reductase: the effect of iron chelators and the role of superoxide in microsomal lipid peroxidation

Superoxide generation, assessed as the rate of acetylated cytochrome c reduction inhibited by superoxide dismutase, by purified NADPH cytochrome P-450 reductase or intact rat liver microsomes was found to account for only a small fraction of their respective NADPH oxidase activities. DTPA-Fe3+ and EDTA-FE3+ greatly stimulated NADPH oxidation, acetylated cytochrome c reduction, and O(2) production by the reductase and intact microsomes. In contrast, all ferric chelates tested caused modest inhibition of acetylated cytochrome c reduction and O(2) generation by xanthine oxidase. Although both EDTA-Fe3+ and DTPA-Fe3+ were directly reduced by the reductase under anaerobic conditions, ADP-Fe3+ was not reduced by the reductase under aerobic or anaerobic conditions. Desferrioxamine-Fe3+ was unique among the chelates tested in that it was a relatively inert iron chelate in these assays, having only minor effects on NADPH oxidation and/or O(2) generation by the purified reductase, intact microsomes, or xanthine oxidase. Desferrioxamine inhibited microsomal lipid peroxidation promoted by ADP-Fe3+ in a concentration-dependent fashion, with complete inhibition occurring at a concentration equal to that of exogenously added ferric iron. The participation of O(2) generated by the reductase in NADPH-dependent lipid peroxidation was also investigated and compared with results obtained with a xanthine oxidase-dependent lipid peroxidation system. NADPH-dependent peroxidation of either phospholipid liposomes or rat liver microsomes in the presence of ADP-Fe3+ was demonstrated to be independent of O(2) generation by the reductase.     

Relation of the structure and function of ferricytochrome c bound to the phosphoprotein phosvitin

The relationship between the structure and function of ferricytochrome c bound to the phosphoprotein phosvitin was investigated. The rates of reduction of phosvitin-bound ferricytochrome c by cytochrome b2, ascorbate and the superoxide radical generated by xanthine oxidase wer repressed where the binding ratio was less than half the maximum, but at higher ratios they were restored gradually with increase in the ratio. The affinity of cytochrome b2 for cytochrome c was not affected by binding of cytochrome c to phosvitin. The redox potential of the bond form was lower than that of the free form and only decreased with decrease in the ratio. The conformatin around the heme moiety and the electronic structure of the heme group of bound ferricytochrome c were similar to those of free ferricytochrome c, but the conformational stability in the vicinity of the prosthetic group was related to the binding ratio as ratios above half the maximum and was well correlated with the reduction rate. Since the binding of cytochrome c to phosvitin is much stronger at binding ratios below half the maximum, these results suggest that this binding strength exclusively affects the conformational flexibility of the heme crevice in the cytochrome molecule, thus altering the reduction rate.     

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.     

The reaction between the superoxide anion radical and cytochrome c.

1. The superoxide anion radical (O2-) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O2- and ferrocytochrome c. 2. At 20 degrees C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4-10(6) M-1. S -1 and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O2- and the form of cytochrome c which exists above pH approximately 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O2- reacts with the form present below pH 7.45 with k = 1.4-10(6) M-1 - S-1, the form above pH 7.45 with k = 3.0- 10(5) M-1 - S-1, and the form present above pH 9.2 with k = 0. 3. The reaction has an activation energy of 20 kJ mol-1 and an enthalpy of activation at 25 degrees C of 18 kJ mol-1 both above and below pH 7.45. It is suggested that O2- may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex. 4. No reduction of ferricytochrome c by HO2 radicals could be demonstrated at pH 1.2-6.2 but at pH 5.3, HO2 radicals oxidize ferrocytochrome c with a rate constant of about 5-10(5)-5-10(6) M-1 - S-1.     

Characterization of the interaction of cytochrome c and mitochondrial ubiquinol-cytochrome c reductase

Characterization of the steady state kinetics of reduction of horse ferricytochrome c by purified beef ubiquinol-cytochrome c reductase, employing 2,3-dimethoxy-5-methyl-6-decylbenzoquinol as reductant, has shown that: 1) the dependence of the reaction on quinol and on ferricytochrome c concentration is consistent with a ping-pong mechanism; 2) the pH optimum of the reaction is near 8.0; 3) the effect of ionic strength on the apparent Km and the TNmax of the reaction for the native cytochrome c is small, and at higher cytochrome c concentrations substrate inhibition is observed; 4) the effect of ionic strength on the kinetic parameters for the reaction of 4-carboxy-2,6-dinitrophenyllysine 27 horse cytochrome c is much larger than for the native protein; and 5) competitive product inhibition is also observed with a Ki consistent with the binding affinity of ferrocytochrome c for Complex III, as determined by gel filtration. In addition, direct binding measurements demonstrated that ferricytochrome c binds more tightly than the reduced protein to Complex III under low ionic strength conditions and that under these conditions more than one molecule of cytochrome c is bound per molecule of Complex III. Exchange of Complex III into a nonionic detergent decreases this excess nonspecific binding. Measurement of the rates of dissociation of the oxidized and reduced 1:1 complexes of cytochrome c and Complex III by stopped flow was consistent with the disparity of binding affinities, the dissociation rate constant for ferrocytochrome c being about 5-fold higher than that for the ferric protein. A model which accounts for the properties of this system is described, assuming that cytochrome c bound to noncatalytic sites on the respiratory complex decreases the catalytic site binding constant for the substrate.     

Flash spectroscopic characterization of photosynthetic electron transport in isolated heterocysts

Electron transport was studied in heterocysts of the filamentous cyanobacterium Anabaena 7120 using spectral and kinetic analysis of absorbance transients elicited by single turnover flashes. Consistent photosynthetic turnovers were observed only in the presence of an exogenous source of reductant; therefore measurements were routinely made under a gas phase containing H2. Prominent absorbance changes corresponding to the oxidation of cytochrome c (554 nm) and the reduction of cytochrome b563 (563 nm) were observed. Under the most reducing conditions (99% H2/1% O2) cytochrome b563 was partially reduced between flashes in a slow, dark reaction. At 10-15% O2, the slow, dark reduction of cytochrome b563 was eliminated. Cytochrome turnover ceased entirely at high O2 concentrations (30%) but was restored by the addition of 25 microM KCN, demonstrating an interaction between the photosynthetic and respiratory electron transfer chains. Strobilurin A slowed the re-reduction of cytochrome c and eliminated the appearance of reduced cytochrome b563 by blocking electron transfer between reduced plastoquinone and the cytochrome b/f complex. Inhibition at a second site was apparent with 2-(n-heptyl)-4-hydroxyquinoline N-oxide, which blocked the reoxidation of cytochrome b563 but had little effect on cytochrome c relaxation. In uncoupled heterocysts, the rates of cytochrome c re-reduction and cytochrome b563 reduction were equal. Additional unassigned absorbance changes at 475 nm, 515 nm, and 572 nm were partially characterized. No absorbance change corresponding to an electrochromic shift was observed.     

Reconstituted microsomal lipid peroxidation: ADP-Fe3+-dependent peroxidation of phospholipid vesicles containing NADPH-cytochrome P450 reductase and cytochrome P450

A reconstituted lipid peroxidation system consisting of rat liver microsomal NADPH-cytochrome P450 reductase and cytochrome P450 incorporated into phospholipid vesicles was developed and characterized. Peroxidation of the vesicles required NADPH and ADP-Fe3+, just as in the NADPH-dependent peroxidation of microsomes. The peroxidation of the vesicles was inhibited 30-50% by superoxide dismutase, depending upon their cytochrome P450 content: those with higher cytochrome P450 contents exhibited greater rates of malondialdehyde formation which were less sensitive to inhibition by superoxide dismutase. When cytochrome P450 was incorporated into vesicles, EDTA-Fe3+ was not required for lipid peroxidation, distinguishing this system from the one previously described by Pederson and Aust [Biochem. Biophys. Res. Comm. 48, 789; 1972]. Since at least 50% of the malondialdehyde formation in the vesicular system was not inhibited by superoxide dismutase, alternative means of iron reduction (O2-.-independent) were examined. It was found that rat liver microsomes or a reconstituted mixed function oxidase system consisting of NADPH-cytochrome P450 reductase and cytochrome P450 in dilauroylphosphatidylcholine micelles reduced ADP-Fe3+ under anaerobic conditions.     

Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.     

Stoichiometry of microsomal oxidation reactions. Distribution of redox-equivalents in monooxygenase and oxidase reactions catalyzed by cytochrome P-450

The stoichiometry of NADPH oxidation in rabbit liver microsomes was studied. It was shown that in uncoupled reactions cytochrome P-450, besides O2- generation catalyzes direct two- and four-electron reduction of O2 to produce H2O2 and water, respectively. With an increase in pH and ionic strength, the amount of O2 reduced via an one-electron route increases at the expense of the two-electron reaction. In parallel, with a rise in pH the steady-state concentration of the oxy-complex of cytochrome P-450 increases, while the synergism of NADPH and NADH action in the H2O2 formation reaction is replaced by competition. The four-electron reduction is markedly accelerated and becomes the main pathway of O2 reduction in the presence of a pseudo-substrate--perfluorohexane. Treatment of rabbit with phenobarbital, which induces the cytochrome P-450 isozyme specific to benzphetamine results in a 2-fold increase in the degree of coupling of NADPH and benzphetamine oxidation. The experimental results suggest that the ratio of reactions of one- and two-electron reduction of O2 is controlled by the ratio of rates of one- and two-electron reduction of cytochrome P-450. In the presence of pseudo-substrates cytochrome P-450 acts predominantly as a four-electron oxidase; one of possible reasons for the uncoupling of microsomal monooxygenase reactions is the multiplicity of cytochrome P-450 isozymes.     

The dioxygen cycle. Spectral, kinetic, and thermodynamic characteristics of ferryl and peroxy intermediates observed by reversal of the cytochrome oxidase reaction.

The catalytic mechanism of O2 reduction by cytochrome oxidase was studied in isolated mitochondria and mitoplasts by partial reversal of the reaction. At a high redox potential (Eh) of cytochrome c, high pH, and a high electrochemical proton gradient (delta mu H+) across the inner mitochondrial membrane, the initial ferriccupric state (O) of the oxidized enzyme's bimetallic oxygen reaction center is converted to ferryl (F) and peroxy (P) intermediates, the optical spectroscopic properties of which are reported in detail. This is associated with reversed electron transfer from the bimetallic center to ferricytochrome c. The kinetics of reduction of ferricytochrome c by the reversed electron transfer process are compared with the kinetics of formation of F and P. The results are consistent with transfer of one electron from the ferric-cupric bimetallic center (O) to cytochrome c, yielding the F intermediate, followed by transfer of one electron from the latter to cytochrome c, yielding the P state. In the absence of an effective redox buffer, poising cytochrome c highly oxidized, these primary events are immediately followed by reoxidation of cytochrome c, which is ascribed to forward electron transfer to enzyme molecules still in the O state. This forward reaction also results in accumulation of the P intermediate. Kinetic stimulations of the data predict equilibrium constants for the reversed electron transfer steps, and Em,7 values of approximately 1.1 and 1.2 V may be calculated for the F/O and P/F redox couples, respectively, at delta mu H+ and delta psi equal to zero. Taken together with previously measured Em,7 values, these data indicate that it is the two-electron reduction of bound dioxygen to bound peroxide that is responsible for the irreversibility of the catalytic dioxygen cycle of cell respiration.     

Modification of the structural and redox properties of cytochrome c by heteropolytungstate binding

Complex formation between horse heart ferricytochrome c and large three-dimensional polyanions has been investigated, in order to study the influence of surface electrostatic interactions on the structural and redox properties of cytochrome c. Cytochrome c binds the large heteropolytungstates (NaSb9W21O86)18- and (KAs4W40O140)27- with a 1/1 polyanion/cytochrome c ratio, and the smaller ion (SiW11O39)8- with a 2/1 ratio. Upon complexation, cytochrome c undergoes structural changes that are dependent on the size and charge of the polyanion, and on the pH and ionic strength of the medium. Three different forms of complexed cytochrome c have been characterized by optical and EPR spectroscopies, in the pH range 6.5-8: an N form, close to the native structure, an A form, analogous to cytochrome c in acidic medium, and a novel B form in which the heme pocket is open but the iron remains low-spin. The redox potential of cytochrome c is lowered to 250-220 mV (vs. NHE) in the N form, and to 80 mV in the B form.     

The effect of complex formation upon the reduction rates of cytochrome c and cytochrome c peroxidase compound II

The effect of complex formation between ferricytochrome c and cytochrome c peroxidase (Ferrocytochrome-c:hydrogen peroxide oxidoreductase, EC 1.11.1.5) on the reduction of cytochrome c by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), reduced N-methylphenazonium methosulfate (PMSH), and ascorbate has been determined at low ionic strength (pH 7) and 25 degrees C. Complex formation with the peroxidase enhances the rate of ferricytochrome c reduction by the neutral reductants TMPD and PMSH. Under all experimental conditions investigated, complex formation with cytochrome c peroxidase inhibits the ascorbate reduction of ferricytochrome c. This inhibition is due to the unfavorable electrostatic interactions between the ascorbate dianion and the negatively charged cytochrome c-cytochrome c peroxidase complex. Corrections for the electrostatic term by extrapolating the data to infinite ionic strength suggest that ascorbate can reduce cytochrome c peroxidase-bound cytochrome c faster than free cytochrome c. Reduction of cytochrome c peroxidase Compound II by dicyanobis(1,10-phenanthroline)iron(II) (Fe(phen)2(CN)2) is essentially unaffected by complex formation between the enzyme and ferricytochrome c at low ionic strength (pH 6) and 25 degrees C. However, reduction of Compound II by the negatively changed tetracyano-(1,10-phenanthroline)iron(II) (Fe(phen)(CN)4) is enhanced in the presence of ferricytochrome c. This enhancement is due to the more favorable electrostatic interactions between the reductant and cytochrome c-cytochrome c peroxidase Compound II complex then for Compound II itself. These studies indicate that complex formation between cytochrome c and cytochrome c peroxidase does not sterically block the electron-transfer pathways from these small nonphysiological reductants to the hemes in these two proteins.