RNA oxidation catalyzed by cytochrome c leads to its depurination and cross-linking, which may facilitate cytochrome c release from mitochondria

Growing evidence indicates that RNA oxidation is correlated with a number of age-related neurodegenerative diseases, and RNA oxidation has also been shown to induce dysfunction in protein synthesis. Here we study in vitro RNA oxidation catalyzed by cytochrome c (cyt c)/H(2)O(2) or by the Fe(II)/ascorbate/H(2)O(2) system. Our results reveal that the products of RNA oxidation vary with the oxidant used. Guanosine residues are preferentially oxidized by cyt c/H(2)O(2) relative to the Fe(II)/ascorbate/H(2)O(2) system. GC/MS and LC/MS analyses demonstrated that the guanine base was not only oxidized but also depurinated to form an abasic sugar moiety. Results from gel electrophoresis and HPLC analyses show that RNA formed a cross-linked complex with cyt c in an H(2)O(2) concentration-dependent manner. Furthermore, when cyt c was associated with liposomes composed of cardiolipin/phosphatidylcholine, and incubated with RNA and H(2)O(2), it was found cross-linked with the oxidized RNA and dissociated from the liposome. Results of the quantitative analysis indicate that the release of the cyt c from the liposome is facilitated by the formation of an RNA-cyt c cross-linked complex. Thus, RNA oxidation may facilitate the release of cyt c from the mitochondrial membrane to induce apoptosis in response to oxidative stress.     

Cytochrome c redox state influences the binding and release of cytochrome c in model membranes and in brain mitochondria

Cytochrome c (cyt c), a component of the respiratory chain, promotes apoptosis when released into the cytosol. Cyt c anchorage within mitochondria depends on cardiolipin (CL). Detachment and release have been related to CL loss and peroxidation. We report that NaN₃-dependent complex IV inhibition, accompanied by impairment of respiration, resulted in cyt c release. Contrarily, inhibition of respiration upstream cyt c with complex I and III inhibitors was not accompanied by the release of the protein, despite CL decrease and monolyso-CL increase. No CL changes and H₂O₂ formation were observed by inhibiting complex IV. In cyt c–CL liposomes, breaching cyt c–CL hydrophilic interactions produced a higher release of the reduced, compared to the oxidized form, suggesting that the hydrophobic component of cyt c–CL binding is prevalent in the oxidized form. Free or liposome-reconstituted cyt c was able to form fatty acid–protein complexes (palmitate < linoleate < oleate) only in its reduced form. We hypothesize that reduced cyt c–fatty acid binding favors the dislocation of the protein from anchoring CL. A mechanism for cyt c release independent of CL peroxidation by H₂O₂ is feasible. It could weaken the hydrophobic component of cyt c–CL interactions and might function following complex IV inhibition or in oxygen lack, both conditions producing accumulation of reduced cyt c and free fatty acids.     

Product-controlled steady-state kinetics between cytochrome aa(3) from Rhodobacter sphaeroides and equine ferrocytochrome c analyzed by a novel spectrophotometric approach

Cytochrome c oxidase (CcO) catalyzes the reduction of molecular oxygen to water using ferrocytochrome c (cyt c(2+)) as the electron donor. In this study, the oxidation of horse cyt c(2+) by CcO from Rhodobacter sphaeroides, was monitored using stopped-flow spectrophotometry. A novel analytic procedure was applied in which the spectra were deconvoluted into the reduced and oxidized forms of cyt c by a least-squares fitting method, yielding the reaction rates at various concentrations of cyt c(2+) and cyt c(3+). This allowed an analysis of the effects of cyt c(3+) on the steady-state kinetics between CcO and cyt c(2+). The results show that cyt c(3+) exhibits product inhibition by two mechanisms: competition with cyt c(2+) at the catalytic site and, in addition, an interaction at a second site which further modulates the reaction of cyt c(2+) at the catalytic site. These results are generally consistent with previous reports, indicating the reliability of the new procedure. We also find that a 6×His-tag at the C-terminus of the subunit II of CcO affects the binding of cyt c at both sites. The approach presented here should be generally useful in spectrophotometric studies of complex enzyme kinetics. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Protein oxidation of cytochrome C by reactive halogen species enhances its peroxidase activity

Reactive halogen species (RHS; X(2) and HOX, where X represents Cl, Br, or I) are metabolites mediated by neutrophil activation and its accompanying respiratory burst. We have investigated the interaction between RHS and mitochondrial cytochrome c (cyt c) by using electrospray mass spectrometry and electron spin resonance (ESR). When the purified cyt c was reacted with an excess amount of hypochlorous acid (HOCl) at pH 7.4, the peroxidase activity of cyt c was increased by 4.5-, 6.9-, and 8.6-fold at molar ratios (HOCl/cyt c) of 2, 4, and 8, respectively. In comparison with native cyt c, the mass spectra obtained from the HOCl-treated cyt c revealed that oxygen is covalently incorporated into the protein as indicated by molecular ions of m/z = 12,360 (cyt c), 12,376 (cyt c + O), and 12,392 (cyt c + 2O). Using tandem mass spectrometry, a peptide (obtained from the tryptic digests of HOCl-treated cyt c) corresponding to the amino acid sequence MIFAGIK, which contains the methionine that binds to the heme, was identified to be involved in the oxygen incorporation. The location of the oxygen incorporation was unequivocally determined to be the methionine residue, suggesting that the oxidation of heme ligand (Met-80) by HOCl results in the enhancement of peroxidase activity of cyt c. ESR spectroscopy of HOCl-oxidized cyt c, when reacted with H(2)O(2) in the presence of the nitroso spin trap 2-methyl-2-nitrosopropane (MNP), yielded more immobilized MNP/tyrosyl adduct than native cyt c. In the presence of H(2)O(2), the peroxidase activity of HOCl-oxidized cyt c exhibited an increasing ability to oxidize tyrosine to tyrosyl radical as measured directly by fast flow ESR. Titration of both native cyt c and HOCl-oxidized cyt c with various amounts of H(2)O(2) indicated that the latter has a decreased apparent K(m) for H(2)O(2), implicating that protein oxidation of cyt c increases its accessibility to H(2)O(2). HOCl-oxidized cyt c also displayed an impaired ability to support oxygen consumption by the purified mitochondrial cytochrome c oxidase, suggesting that protein oxidation of cyt c may break the electron transport chain and inhibit energy transduction in mitochondria.     

Cytochrome c(553), a small heme protein that lacks misligation in its unfolded state, folds with rapid two-state kinetics

Cytochrome c(553) (cyt c(553)) from Desulfovibrio vulgaris is a small helical heme protein that displays apparent two-state equilibrium-unfolding behavior. The covalently attached heme is low-spin, ligated by Met and His residues, in the native state but becomes high-spin upon unfolding at pH 7. Here, we show that in contrast to other c-type heme proteins, where misligations in the unfolded states are prominent, cyt c(553) refolding kinetics at pH 7 proceeds rapidly without detectable intermediates. The extrapolated folding rate constant in water for oxidized cyt c(553) matches exactly that predicted from the cyt c(553) native-state topology: 5300 s(-1 )(experimental) versus 5020 s(-1) (predicted). We therefore conclude that the presence of the oxidized cofactor does not affect the intrinsic formation speed of the cyt c(553 )structural motif.     

Biochemical properties of cytochrome c nitrated by peroxynitrite

Nitration of tyrosine residues is taken as evidence for intracellular formation of peroxynitrite. Cytochrome c (cyt c) can be nitrated by peroxynitrite and nitrated cyt c has been observed in cells and tissues under stress conditions. Here we studied the biochemical properties of nitrated cyt c in order to understand its potential roles in nitrative stress. Nitration of cyt c resulted in disruption of the heme-methionine bond and rapid binding to cyanide. Equilibrium unfolding by guanidine hydrochloride showed that cyt c was slightly destabilized upon nitration but the unfolding transition of nitrated cyt c was highly cooperative indicating that the overall folding was largely preserved. Nitrated cyt c could not be reduced by superoxide and did not support electron transfer between ascorbate and cyt c oxidase. Nitration of cyt c resulted in a tremendous increase in peroxidase activity so that nitrated cyt c rapidly oxidized dihydrodichlorofluorescein even in the presence of a high concentration of glutathione. Enhanced peroxidase activity of nitrated cyt c was responsible for H2O2-induced oxidation of phospholipid membranes and H2O2/NO2--mediated nitration of other proteins. These results suggest that nitration of cyt c by peroxynitrite may exacerbate oxidative damage to mitochondrial proteins and membranes.     

Effect of famoxadone on photoinduced electron transfer between the iron-sulfur center and cytochrome c1 in the cytochrome bc1 complex

Famoxadone is a new cytochrome bc(1) Q(o) site inhibitor that immobilizes the iron-sulfur protein (ISP) in the b conformation. The effects of famoxadone on electron transfer between the iron-sulfur center (2Fe-2S) and cyt c(1) were studied using a ruthenium dimer to photoinitiate the reaction. The rate constant for electron transfer in the forward direction from 2Fe-2S to cyt c(1) was found to be 16,000 s(-1) in bovine cyt bc(1). Binding famoxadone decreased this rate constant to 1,480 s(-1), consistent with a decrease in mobility of the ISP. Reverse electron transfer from cyt c(1) to 2Fe-2S was found to be biphasic in bovine cyt bc(1) with rate constants of 90,000 and 7,300 s(-1). In the presence of famoxadone, reverse electron transfer was monophasic with a rate constant of 1,420 s(-1). It appears that the rate constants for the release of the oxidized and reduced ISP from the b conformation are the same in the presence of famoxadone. The effects of famoxadone binding on electron transfer were also studied in a series of Rhodobacter sphaeroides cyt bc(1) mutants involving residues at the interface between the Rieske protein and cyt c(1) and/or cyt b.     

H(2)O(2) disposal in cardiolipin-enriched brain mitochondria is due to increased cytochrome c peroxidase activity

The mitochondrial electron transport chain is a source of oxygen superoxide anion (O(2)(-)) that is dismutated to H(2)O(2). Although low levels of ROS are physiologically synthesized during respiration, their increase contributes to cell injury. Therefore, an efficient machinery for H(2)O(2) disposal is essential in mitochondria. In this study, the ability of brain mitochondria to acquire cardiolipin (CL), phosphatidylglycerol (PG), and phosphatidylserine (PS) in vitro through a fusion process was exploited to investigate lipid effects on ROS. MTT assay, oxygen consumption, and respiratory ratio indicated that the acquired phospholipids did not alter mitochondrial respiration and O(2)(-) production from succinate. However, in CL-enriched mitochondria, H(2)O(2) levels where 27% and 47% of control in the absence and in the presence of antimycin A, respectively, suggesting an increase in H(2)O(2) elimination. Concomitantly, cytochrome c (cyt c) was released outside mitochondria. Since free oxidized cyt c acquired peroxidase activity towards H(2)O(2) upon interaction with CL in vitro, a contribution of cyt c to H(2)O(2) disposal in mitochondria through CL conferred peroxidase activity is plausible. In this model, the accompanying CL peroxidation should weaken cyt c-CL interactions, favouring the detachment and release of the protein. Neither cyt c peroxidase activity was elicited by PS in vitro, nor cyt c release was observed in PS-enriched mitochondria, although H(2)O(2) levels were significantly decreased, suggesting a cyt c-independent role of PS in ROS metabolism in mitochondria.     

Surface-enhanced resonance Raman spectroscopy and spectroscopy study of redox-induced conformational equilibrium of cytochrome c adsorbed on DNA-modified metal electrode

The redox-induced conformational equilibrium of cytochrome c (cyt c) adsorbed on DNA-modified metal electrode and the interaction mechanism of DNA with cyt c have been studied by electrochemical, spectroscopic and spectroelectrochemical techniques. The results indicate that the external electric field induces potential-dependent coordination equilibrium of the adsorbed cyt c between its oxidized state (with native six-coordinate low-spin and non-native five-coordinate high-spin heme configuration) and its reduced state (with native six-coordinate low-spin heme configuration) on DNA-modified metal electrode. The strong interactions between DNA and cyt c induce the self-aggregation of cyt c adsorbed on DNA. The orientational distribution of cyt c adsorbed on DNA-modified metal electrode is potential-dependent, which results in the deviation from an ideal Nernstian behavior of the adsorbed cyt c at high electrode potentials. The electric-field-induced increase in the activation barrier of proton-transfer steps attributed to the rearrangement of the hydrogen bond network and the self-aggregation of cyt c upon adsorption on DNA-modified electrode strongly decrease the interfacial electron transfer rate. In addition, the strongly Coulombic interactions between DNA and cyt c only disturb the microenvironment of the heme, and do not affect the states of heme ligation and spin. The secondary structure of the adsorbed cyt c is retained, while the conformation of DNA is changed from the B form DNA to A form DNA.     

Surface properties of monomolecular films of oxidized and reduced cytochrome c and f.

The surface properties of monomolecular films of oxidized and reduced cytochromes f and c were measured at an air-water interface. Area/molecular (A) and surface potential (deltaV) for oxidized and reduced forms of the cytochromes were measured as a function of pH. Oxidized cyt f has a maximum for both A and deltaV at pH 7.5. At a surface pressure of 6 dyn/cm the maximum A equals 2600 plus or minus 50 A2 and the maximum deltaV equals 200 plus or minus 10 mV. Reduced cyt f as a function of pH has a minimum value for both A (2200 A2) and deltaV (95 mV). Oxidized cyt c as a function of pH has minima for A (140 A2) and deltaV (188 mV) at pH 7.0 and 7.3, respectively. On the other hand, reduced cyt has maximum values for A (220 A2) and deltaV (260 mV) at pH 7.0 and 7.3, respectively.     

Marked difference in cytochrome c oxidation mediated by HO(*) and/or O(2)(*-) free radicals in vitro

Cytochrome c (cyt c) is an electron carrier involved in the mitochondrial respiratory chain and a critical protein in apoptosis. The oxidation of cytochrome c can therefore be relevant biologically. We studied whether cytochrome c underwent the attack of reactive oxygen species (ROS) during ionizing irradiation-induced oxidative stress. ROS were generated via water radiolysis under ionizing radiation (IR) in vitro. Characterization of oxidation was performed by mass spectrometry, after tryptic digestion, and UV-visible spectrophotometry. When both hydroxyl and superoxide free radicals were generated during water radiolysis, only five tryptic peptides of cyt c were reproducibly identified as oxidized according to a relation that was dependent of the dose of ionizing radiation. The same behavior was observed when hydroxyl free radicals were specifically generated (N(2)O-saturated solutions). Specific oxidation of cyt c by superoxide free radicals was performed and has shown that only one oxidized peptide (MIFAGIK+16), corresponding to the oxidation of Met80 into methionine sulfoxide, exhibited a radiation dose-dependent formation. In addition, the enzymatic site of cytochrome c was sensitive to the attack of both superoxide and hydroxyl radicals as observed through the reduction of Fe(3+), the degradation of the protoporphyrin IX and the oxidative disruption of the Met80-Fe(3+) bond. Noteworthy, the latter has been involved in the conversion of cyt c to a peroxidase. Finally, Met80 appears as the most sensitive residue towards hydroxyl but also superoxide free radicals mediated oxidation.     

Characterization of the reaction products of cytochrome c with glutathione by mass spectrometry

Cytochrome c and glutathione (GSH) are two important biomolecules that regulate many cellular processes. The reaction of cytochrome c with GSH involves radical oxygen species and exhibits significant complexity. In the present work, the reaction of cytochrome c with GSH in water was characterized using mass spectrometry. The results show for the first time that the reaction generates multiple products including apocytochrome c in oxidized and reduced forms, glutathionylated apocytochrome c, GSH-modified cytochrome c, and oxidized and hydroxylated species. The reaction is O(2) dependent and is rapid in water at neutral pH and 37 degrees C. The reaction involves the cleavage of thioether linkages between the heme and apocytochrome c. Evidence for the role of H(2)O(2) and other oxygen radicals in this reaction is also provided.     

Supramolecular organisation of the photosynthetic chain in anoxygenic bacteria

This minireview summarizes our present view of the supramolecular organization of the photosynthetic apparatus of Rhodobacter sphaeroides and Rhodobacter capsulatus. These two species present a close association between two reaction centers (RCs), one cytochrome (cyt) bc(1) and one cyt c. In R. sphaeroides, the RCs are only partially surrounded by LH1 complexes. This open ring of LH1 complexes is required for an efficient photoinduced cyclic electron transfer only under conditions where the quinone pool totally reduced. When the quinone pool is partially oxidized, a closed ring of LH1 complexes around the RCs does not impair the exchange of quinone molecules between the RC and the cyt bc(1) complex. To explain the efficient photochemistry of the various species which possess a RC surrounded by a closed ring of LH, it is proposed that their quinone pool is partially oxidized even under anaerobic condition.     

Axial ligand replacement in horse heart cytochrome c by semisynthesis

Semisynthesis has been employed to replace the axial methionine in horse heart cytochrome c with histidine. The reduction potential of the His-80 protein (cyt c-His-80) is 41 mV vs NHE (0.1 M phosphate; pH 7.0; 25 degrees C). The absorption spectra of oxidized and reduced cyt c-His-80 are very similar to those of the native protein in the porphyrin region, but the 695 nm band is absent in the oxidized His-80 protein.     

The "pro-apoptotic genies" get out of mitochondria: oxidative lipidomics and redox activity of cytochrome c/cardiolipin complexes

One of the prominent consequences of the symbiogenic origin of eukaryotic cells is the unique presence of one particular class of phospholipids, cardiolipin (CL), in mitochondria. As the product originated from the evolution of symbiotic bacteria, CL is predominantly confined to the inner mitochondrial membrane in normally functioning cells. Recent findings identified CL and its oxidation products as important participants and signaling molecules in the apoptotic cell death program. Early in apoptosis, massive membrane translocations of CL take place resulting in its appearance in the outer mitochondrial membrane. Consequently, significant amounts of CL become available for the interactions with cyt c, one of the major proteins of the intermembrane space. Binding of CL with cytochrome c (cyt c) yields the cyt c/CL complex that acts as a potent CL-specific peroxidase and generates CL hydroperoxides. In this review, we discuss the catalytic mechanisms of CL oxidation by the peroxidase activity of cyt c as well as the role of oxidized CL (CLox) in the release of pro-apoptotic factors from mitochondria into the cytosol. Potential implications of cyt c/CL peroxidase intracellular complexes in disease conditions (cancer, neurodegeneration) are also considered. The discovery of the new role of cyt c/CL complexes in early mitochondrial apoptosis offers interesting opportunities for new targets in drug discovery programs. Finally, exit of cyt c from damaged and/or dying (apoptotic) cells into extracellular compartments and its accumulation in biofluids is discussed in lieu of the formation of its peroxidase complexes with negatively charged lipids and their significance in the development of systemic oxidative stress in circulation.     

Photooxidation of cytochrome b559 in oxygen-evolving photosystem II.

Cytochrome b559 (cyt b559) is an intrinsic and essential component of the photosystem II (PSII) protein complex, but its function, stoichiometry, and electron-transfer kinetics in the physiological system are not well-defined. In this study, we have used flash-detection optical spectroscopy to measure the kinetics and yields of photooxidation and dark reduction of cyt b559 in untreated, O2-evolving PSII-enriched membranes at room temperature. The dark redox states of cyt b559 and the primary electron acceptor, QA, were determined over the pH range 5.0-8.5. Both the fraction of dark-oxidized cyt b559 and dark-reduced QA increased with increasing acidity. Consistent with these results, an acid-induced drop in pH from 8.5 to 4.9 in a dark-adapted sample caused the oxidation of cyt b559, indicating a shift in the redox state during the dark reequilibration. As expected from the dark redox state of cyt b559, the rate and extent of photooxidation of cyt b559 during continuous illumination decreased toward more acidic pH values. After a single, saturating flash, the rate of photooxidation of cyt b559 was of the same order of magnitude as the rate of S2QA- charge recombination. In untreated PSII samples at pH 8.0 with 42% of cyt b559 oxidized and 15% of QA reduced in the dark, 4.7% of one copy of cyt b559 was photooxidized after one flash with a t1/2 of 540 +/- 90 ms. On the basis of our previous work [Buser, C. A., Thompson, L. K., Diner, B. A., & Brudvig, G. W (1990) Biochemistry 29, 8977] and the data presented here, we conclude that Sn+1, YZ., and P680+ are in redox equilibrium and cyt b559 (and YD) are oxidized via P680+. After a period of illumination sufficient to fully reduce the plastoquinone pool, we also observed the pH-dependent dark reduction of photooxidized cyt b559, where the rate of reduction decreased with decreasing pH and was not observed at pH < 6.4. To determine the direct source of reductant to oxidized cyt b559, we studied the dark reduction of cyt b559 and the reduction of the PQ pool as a function of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) concentration. We find that DCMU inhibits the reduction of cyt b559 under conditions where the plastoquinone pool and QA are reduced. We conclude that QB-. (H+) or QBH2 is the most likely source of the electron required for the reduction of oxidized cyt b559.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytochrome b5 inhibits electron transfer from NADPH-cytochrome P450 reductase to ferric cytochrome P450 2B4

Experiments demonstrating that cytochrome (cyt) b5 inhibits the activity of cytochrome P450 2B4 (cyt P450 2B4) at higher concentrations suggested that cyt b5 was occupying the cyt P450 reductase-binding site on cyt P450 2B4 and preventing the reduction of ferric cyt P450 (Zhang, H., Im, S.-C., and Waskell, L. (2007) J. Biol. Chem. 282, 29766-29776). In this work experiments were undertaken with manganese-containing cyt b5 (Mn-cyt b5) to test this hypothesis. Because Mn-cyt b5 does not undergo oxidation state changes under our experimental conditions, interpretation of the experimental results was unambiguous. The rate of electron transfer from cyt P450 reductase to ferric cyt P450 2B4 was decreased by Mn-cyt b5 in a concentration-dependent manner. Moreover, reduction of cyt P450 2B4 by cyt P450 reductase was incomplete in the presence of Mn-cyt b5. At a Mn-cyt b(5):cyt P450 2B4:cyt P450 reductase molar ratio of 5:1:1, the rate of reduction of ferric cyt P450 was decreased by 10-fold, and only 30% of the cyt P450 was reduced, whereas 70% remained oxidized. It could be demonstrated that Mn-cyt b5 had its effect by acting on cyt P450, not the reductase, because the reduction of cyt c by cyt P450 reductase in the presence of Mn-cyt b5 was not effected. Furthermore, under steady-state conditions in the cyt P450 reconstituted system, Mn-cyt b5, which lacks the ability to reduce oxyferrous cyt P450 2B4, was unable to stimulate the activity of cyt P450. Mn-cyt b5 only inhibited the cyt P450 2B4 activity. In conjunction with site-directed mutagenesis studies and experiments that strongly suggested that cyt b5 competed with cyt P450 reductase for binding to cyt P450, the current investigation demonstrates unequivocally that cyt b5 inhibits the activity of cyt P450 2B4 by preventing cyt P450 reductase from binding to cyt P450, a prerequisite for electron transfer from cyt P450 reductase to cyt P450 and catalysis.     

Domain conformational switch of the iron-sulfur protein in cytochrome bc1 complex is induced by the electron transfer from cytochrome bL to bH

Intensive biochemical, biophysical and structural studies of the cytochrome (cyt) bc(1) complex in the past have led to the formulation of the "protonmotive Q-cycle" mechanism for electron and proton transfer in this vitally important complex. The key step of this mechanism is the separation of electrons during the oxidation of a substrate quinol at the Q(P) site with both electrons transferred simultaneously to ISP and cyt b(L) when the extrinsic domain of ISP (ISP-ED) is located at the b-position. Pre-steady state fast kinetic analysis of bc(1) demonstrates that the reduced ISP-ED moves to the c(1)-position to reduce cyt c(1) only after the reduced cyt b(L) is oxidized by cyt b(H). However, the question of how the conformational switch of ISP-ED is initiated remains unanswered. The results obtained from analysis of inhibitory efficacy and binding affinity of two types of Q(P) site inhibitors, Pm and Pf, under various redox states of the bc(1) complex, suggest that the electron transfer from heme b(L) to b(H) is the driving force for the releasing of the reduced ISP-ED from the b-position to c(1)-position to reduce cyt c(1).