Mitochondrial regulation of caspase activation by cytochrome oxidase and tetramethylphenylenediamine via cytosolic cytochrome c redox state

Cytochrome c release from mitochondria induces caspase activation in cytosols; however, it is unclear whether the redox state of cytosolic cytochrome c can regulate caspase activation. By using cytosol isolated from mammalian cells, we find that oxidation of cytochrome c by added cytochrome oxidase stimulates caspase activation, whereas reduction of cytochrome c by added tetramethylphenylenediamine (TMPD) or yeast lactate dehydrogenase/cytochrome c reductase blocks caspase activation. Scrape-loading of cells with this reductase inhibited caspase activation induced by staurosporine. Similarly, incubating intact cells with ascorbate plus TMPD to reduce intracellular cytochrome c strongly inhibited staurosporine-induced cell death, apoptosis, and caspase activation but not cytochrome c release, indicating that cytochrome c redox state can regulate caspase activation. In homogenates from healthy cells cytochrome c was rapidly reduced, whereas in homogenates from apoptotic cells added cytochrome c was rapidly oxidized by some endogenous process. This oxidation was prevented if mitochondria were removed from the homogenate or if cytochrome oxidase was inhibited by azide. This suggests that permeabilization of the outer mitochondrial membrane during apoptosis functions not just to release cytochrome c but also to maintain it oxidized via cytochrome oxidase, thus maximizing caspase activation. However, this activation can be blocked by adding TMPD, which may have some therapeutic potential.     

Respiratory cytochrome c oxidase can be efficiently reduced by the photosynthetic redox proteins cytochrome c6 and plastocyanin in cyanobacteria

Plastocyanin and cytochrome c6 are two small soluble electron carriers located in the intrathylacoidal space of cyanobacteria. Although their role as electron shuttle between the cytochrome b6f and photosystem I complexes in the photosynthetic pathway is well established, their participation in the respiratory electron transport chain as donors to the terminal oxidase is still under debate. Here, we present the first time-resolved analysis showing that both cytochrome c6 and plastocyanin can be efficiently oxidized by the aa3 type cytochrome c oxidase in Nostoc sp. PCC 7119. The apparent electron transfer rate constants are ca. 250 and 300 s(-1) for cytochrome c6 and plastocyanin, respectively. These constants are 10 times higher than those obtained for the oxidation of horse cytochrome c by the oxidase, in spite of being a reaction thermodynamically more favourable.     

The pH in reversed micelles as imposed by the dihydrogen/proton redox couple and indicated by viologens and cytochrome c3 using hydrogenase as redox catalyst.

The pH values in reversed micelles were measured, making use of the hydrogenase enzyme as redox catalyst short-circuiting the viologen oxidized/semiquinone redox states. The hydrogenases from Desulfovibrio vulgaris (Hildenborough) and from Megasphaera elsdenii were applied. The observed pH values in reversed micelles were not dependent on the type of hydrogenase. Two cationic [cetyltrimethylammonium bromide and dodecylammonium propionate (DAP)] and two anionic sodiumdodecyl sulphate, sodium di(ethylhexyl)sulfosuccinate types of reversed micelles were used in combination with viologens having distinguishable valencies. It was observed that, in the cationic-reversed micelles, the dissociation constant for the semiquinone dimer had about the same value as compared to bulk water, while this value was significantly higher in the anionic-reversed micelles. Furthermore, the dissociation constant was independent of the concentration of viologen semiquinone in the reversed micelle, indicating that exchange kinetics are faster than the dimerisation process. With the exception of DAP, a linear relation exists, pH = a.pHrm + b, between the pH of the bulk water and the pH as measured in the reversed micelle (pHrm). In all these cases the value of a is smaller than unity, the value of b ranges between 1.6-2.7. For DAP the pHrm is always around 7. In DAP-reversed micelles, the counter-ion propionate probably serves as an internal buffer. Using cytochrome c3 as pH indicator in combination with N,N'-di(3-aminopropyl)-4,4'-bipyridinium)4+ to take care of electron transfer, in cetyltrimethylammonium-bromide-reversed micelles the pHrm is about the same as indicated by the viologen; in SDS-reversed micelles the pHrm is always lower than that indicated by N,N'-di(3-aminopropyl)4,4'-pyridinium4+. In contrast to cytochrome c3 from D. vulgaris, which in reversed micelles cannot become reduced directly by its D. vulgaris hydrogenase, the hydrogenase of M. elsdenii is able to reduce its ferredoxin directly.     

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.     

Heme-Cu complexes as oxygen-activating functional models for the active site of cytochrome c oxidase

Tri(2-pyridylmethyl)amineCu complex-linked iron meso-tetraphenylporphyine derivatives were prepared to model the active site of cytochrome c oxidase. Exposure to oxygen converted the reduced forms of the complexes to the corresponding stable mu-peroxo species in spite of the presence of three coordination sites, two on the heme and one on the Cu. The oxy forms were characterized spectroscopically. Kinetic analyses of the oxygenation reactions of the reduced forms suggests that preferential O2 binding occurs at the Cu site over the heme. This mechanism is also supported by examination of the redox potentials of the two metal ions. Since the peroxy complexes of the models exhibit a structure similar to that of the previously reported fully-oxidized form, the relevance of the model chemistry to the enzyme reaction is discussed.     

Influence of vitamin C and vitamin E on redox signaling: Implications for exercise adaptations

The exogenous antioxidants vitamin C (ascorbate) and vitamin E (α-tocopherol) often blunt favorable cell signaling responses to exercise, suggesting that redox signaling contributes to exercise adaptations. Current theories posit that this antioxidant paradigm interferes with redox signaling by attenuating exercise-induced reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. The well-documented in vitro antioxidant actions of ascorbate and α-tocopherol and characterization of the type and source of the ROS/RNS produced during exercise theoretically enable identification of redox-dependent mechanisms responsible for the blunting of favorable cell signaling responses to exercise. This review aimed to apply this reasoning to determine how the aforementioned antioxidants might attenuate exercise-induced ROS/RNS production. The principal outcomes of this analysis are (1) neither antioxidant is likely to attenuate nitric oxide signaling either directly (reaction with nitric oxide) or indirectly (reaction with derivatives, e.g., peroxynitrite); (2) neither antioxidant reacts appreciably with hydrogen peroxide, a key effector of redox signaling; (3) ascorbate but not α-tocopherol has the capacity to attenuate exercise-induced superoxide generation; and (4) alternate mechanisms, namely pro-oxidant side reactions and/or reduction of bioactive oxidized macromolecule adducts, are unlikely to interfere with exercise-induced redox signaling. Out of all the possibilities considered, ascorbate-mediated suppression of superoxide generation with attendant implications for hydrogen peroxide signaling is arguably the most cogent explanation for blunting of favorable cell signaling responses to exercise. However, this mechanism is dependent on ascorbate accumulating at sites rich in NADPH oxidases, principal contributors to contraction-mediated superoxide generation, and outcompeting nitric oxide and superoxide dismutase isoforms. The major conclusions of this review are: (1) direct evidence for interference of ascorbate and α-tocopherol with exercise-induced ROS/RNS production is lacking; (2) theoretical analysis reveals that both antioxidants are unlikely to have a major impact on exercise-induced redox signaling; and (3) it is worth considering alternate redox-independent mechanisms.     

Intermediate and stable redox states of cytochrome c studied by low temperature resonance Raman spectroscopy

Stabilized intermediate redox states of cytochrome c are generated by radiolytic reduction of initially oxidized enzyme in glass matrices at liquid nitrogen temperature. In the intermediate states the heme group is reduced by hydrated electrons, whereas the protein conformation is restrained close to its oxidized form by the low-temperature glass matrix. The intermediate and stable redox states of cytochrome c at neutral and alkaline pH are studied by low-temperature resonance Raman spectroscopy using excitations in resonance with the B (Soret) and Q1 (beta) optical transitions. The assignments of the cytochrome c resonance Raman bands are discussed. The observed spectral characteristics of the intermediate states as well as of the alkaline transition in the oxidized state are interpreted in terms of oxidation-state marker modes, spin-state marker modes, heme iron--axial ligand stretching modes, totally symmetric in-plane porphyrin modes, nontotally symmetric in-plane modes, and out-of-plane modes.     

Redox potentials in hydro-organic media at normal and subzero temperatures. Ferro-ferricyanide and cytochrome c as models.

Redox potentials of ferro-ferricyanide and cytochrome c were measured in water/ethylene glycol and water/dimethylsulfoxide (volume ratio from 100/0 to 50/50) between 25 and -25 degrees C. For both systems, the midpoint potential decreases in the presence of organic solvents and increases by cooling. The magnitude of these variations is larger in dimethylsulfoxide than in ethylene glycol; moreover in the same solvent mixture it is larger with ferro-ferricyanide than with cytochrome c, so that the difference between the redox potentials of these two systems can be strongly affected and even reversed. While in pure water (cacodylate buffer pH 7.0, NaCl 0.1 M) they are respectively +388 and +265 mV, in 50% dimethylsulfoxide at 25 degrees C they decrease to +112 and +208 mV. Reduction of cytochrome c by ferro-ferricyanide, in this mixture, is then expected and was indeed observed. On the other hand, as (deltaE/deltaT)T, (E being the redox potential) is higher for ferro-ferricyanide than for cytochrome c, the oxidative power of the former for the latter is expected to increase as temperature decreases. This effect was observed in 50% ethylene glycol at -16 degrees C. Organic solvents and large temperature variations appear then as powerful perturbants of redox reactions. Their effects should be taken into account in studies of redox reactions carried out in cooled hydro-organic media.     

Cytochrome c and cytochrome c peroxidase complex as studied by resonance Raman spectroscopy.

Complex formation between ferricytochrome c peroxidase (CCP) and ferricytochrome c from yeast [cyt(Y)] and horse heart [cyt(H)] was studied by resonance Raman spectroscopy. On the basis of a detailed spectral analysis of the free proteins, it was possible to attribute changes in the spectra of the complexes to the individual proteins. At pH 7.0 both cyt(Y) and cyt(H) binding induces an increase in the six-coordinate low-spin configuration of CCP from 9% to 19% at the expense of the five-coordinate high-spin state, which drops from 84% to 74%. In the free and complexed state, CCP exhibits a constant fraction of the six-coordinate high-spin form (approximately 7%). In addition to affecting the coordination state, there is also a cyt-specific structural response of CCP to complexation. In the cyt(Y)-CCP complex, the peripheral vinyl and propionate substituents of CCP are more rigidly fixed in the protein matrix, whereas binding of cyt(H) only slightly perturbs the conformations of these side chains. The biological significance of the conformational changes in CCP are discussed. In contrast to CCP, there are no detectable structural changes in either cyt(Y) or cyt(H) upon complex formation.     

Donnan effect as measured by sedimentation equilibrium for the protein cytochrome c.

The protein turkey-heart cytochrome c is used as a model protein to study charge effects in sedimentation equilibrium experiments in three-component solutions. Data are given for the dependence of the apparent M (1–υ ρ) on ρ in solutions of KCl, RbCl, CsCl, and triethylamine hydrochloride. The results show the Donnan effect to have a significant influence on the apparent molecular weight, found by extrapolation of the data to a solution density of one. The apparent molecular weights are for protein at infinite dilution. A theoretical treatment is presented where the magnitude of this effect can be predicted accurately from the formal net charge of the protein as computed from the amino acid composition. The results are shown to be important in computing the preferential hydration of the protein in concentrated salt solutions. For such systems the Donnan effect should be subtracted from the total interaction coefficient for multicomponent system in order to obtain the preferential hydration.     

Conformational isomerism and effective redox geometry in the oxidation of heme proteins by alkyl halides, cytochrome c, and cytochrome oxidase.

In contrast to its lethargy at physiological pH, horse heart cytochrome c can be oxidized at room temperature by the axial inner sphere oxidant bromomalononitrile (BMN) at higher acidities. The following stoichiometry obtains: 2Fe11 c + BrCH(CN2) + H+ leads to 2FeIII c + CH2(CN)2 + Br-, and the rate law is given by: rate = k2(FeIIc)(BMN). At an ionic strength of 1.0 (KCl), second-order rate constants vary from 300 l. per mol per sec (pH 2-3) to 0(pH 9). Below pH 6 there is a noticeable increase in rate with ionic strength while there is no specific salt effect for the process. At pH 7.4 there is no influence of added salt (0.01-1.0 M) upon the slow rate of reaction. The vast changes in rate occur over a pH region (3-6) in which only very minor changes in the visible spectrum of the cytochrome are manifest. The results are interpreted in terms of a conformational isomerism of cytochrome c in which the effective redox geometry alters from a predominantly "short C" form (in which an axial position is available for substitution) at lower pH's to a predominantly "C" form (axial positions encumbered) in the physiological region. At 5 degrees, pH 7.4, both hemes of beef heart cytochrome oxidase are oxidized by the addition of BMN (k2 = 29 plus or minus 3 l. per mol per sec). However, the reaction is inhibited by potassium cyanide and the protein containing iron(II) cyt alpha along with the cyano adduct of iron(II) or iron(III) cyt alpha3 is inert. The results demonstrate cytochrome alpha3 as the site of reaction and that alpha reduces alpha3 in the process. Cytochrome oxidase does catalyze the oxidation of cytochrome c with BMN as substrate. Taken together the results provide additional support for a recent theory and they demonstrate BMN to be an efficient probe for the effective redox geometry of a hemoprotein in solution.     

Kinetic studies on redox reactions of hemoproteins. I. Reduction of thermoresistant cytochrome c-552 and horse heart cytochrome c by ferrocyanide.

The oxidation-reduction reaction of horse heart cytochrome c and cytochrome c (552, Thermus thermophilus), which is highly thermoresistant, was studied by temperature-jump method. Ferrohexacyanide was used as reductant. (Formula: see text.) Thermodynamic and activation parameters of the reaction obtained for both cytochromes were compared with each other. The results of this showed that (1) the redox potential of cytochrome c-552, + 0.19 V, is markedly less than that of horse heart cytochrome c. (2) deltaHox of cytochrome c-552 is considerably lower than that of horse heart cytochrome c. (3) deltaSox and deltaSred of cytochrome c-552 are more negative than those of horse heart cytochrome c. (4) kred of cytochrome c-552 is much lower than that of horse heart cytochrome c at room temperature.     

Separation of enzymically active bovine cytochrome c oxidase monomers and dimers by high performance liquid chromatography

The aggregation state of two types of bovine heart cytochrome c oxidase preparations in the presence of laurylmaltoside was investigated by high performance liquid chromatography in two buffers of ionic strengths of 388 mM and 45 mM, respectively. At high ionic strength, it was found that the Fowler cytochrome c oxidase preparation was monomeric (Mr = 2 X 10(5)), while monomers and dimers (2 X aa3, Mr = 4 X 10(5)) could be isolated from the Yonetani preparation. Under these conditions there was no rapid equilibrium between the two forms. Covalent cytochrome c oxidase-cytochrome c complexes were largely dimeric, and addition of ascorbate and cytochrome c to the oxidase also promoted dimerization. At low ionic strength (I = 45 mM) in the presence of laurylmaltoside the oxidase and the covalent complex with cytochrome c were largely monomeric. In the steady-state oxidation of ferrous horse heart cytochrome c, the monomeric enzyme displayed biphasic kinetics at I = 45 mM. This suggests that the presence of high- and low-affinity reactions is an intrinsic property of the cytochrome c oxidase monomer.     

Inhibition of plant and animal cytochrome oxidases by nitrous oxide as a function of cytochrome c concentration.

Cytochrome oxidase from both pea leaves and bovine heart shows lower activity under a mixture of 79% N2O/21% O2 than under ambient air. This inhibition is not detectable below 5 microM cytochrome c but appears with increasing concentrations of cytochrome c. These results suggest that the N2O-induced inhibition of cytochrome c oxidase is modulated by cytochrome c concentration. This seems to concern only the lowest affinity site of the oxidase. Apparently, N2O and cytochrome c do not share the same site of fixation on the oxidase.     

Kinetic studies on redox reactions of hemoproteins. II. Reduction of thermoresistant cytochrome c-552 and horse heart cytochrome c by ascorbic acid.

The reductions of thermoresistant cytochrome c-552 and horse heart cytochrome c by ascorbic acid were studied by the stopped-flow method between pH 4 and 10. The results were as follows (1) The reduction of horse heart cytochrome c showed two relaxation decays above pH 8.5, one of which was pseudo-first order, as was the case below pH 8, while the other was nearly concentration-independent. These results were consistent with those reported by Greenwood and Palmer (J. Biol. Chem. (1965) 240, 3660-3663). (2) For the reduction of cytochrome c-552, only a single relaxational decay that obeyed pseudo-first order kinetics was observed. (3) It seems most reasonable to assume that the concentration-independent relaxation process can be attributed to the isomerization reaction accompanying ligand exchange, since it is known that only horse heart cytochrome c exhibits ligand exchange, involving a residue with pK 9.3.     

Crystal structure of human SCO1: implications for redox signaling by a mitochondrial cytochrome c oxidase "assembly" protein

Human SCO1 and SCO2 are copper-binding proteins involved in the assembly of mitochondrial cytochrome c oxidase (COX). We have determined the crystal structure of the conserved, intermembrane space core portion of apo-hSCO1 to 2.8 A. It is similar to redox active proteins, including thioredoxins (Trx) and peroxiredoxins (Prx), with putative copper-binding ligands located at the same positions as the conserved catalytic residues in Trx and Prx. SCO1 does not have disulfide isomerization or peroxidase activity, but both hSCO1 and a sco1 null in yeast show extreme sensitivity to hydrogen peroxide. Of the six missense mutations in SCO1 and SCO2 associated with fatal mitochondrial disorders, one lies in a highly conserved exposed surface away from the copper-binding region, suggesting that this region is involved in protein-protein interactions. These data suggests that SCO functions not as a COX copper chaperone, but rather as a mitochondrial redox signaling molecule.