The role of tryptophan 272 in the Paracoccus denitrificans cytochrome c oxidase

The mechanism of electron coupled proton transfer in cytochrome c oxidase (CcO) is still poorly understood. The P(M)-intermediate of the catalytic cycle is an oxoferryl state whose generation requires one additional electron, which cannot be provided by the two metal centres. The missing electron has been suggested to be donated to this binuclear site by a tyrosine residue. A tyrosine radical species has been detected in the P(M) and F* intermediates (formed by addition of H2O2) of the Paraccocus denitrificans CcO using electron paramagnetic resonance (EPR) spectroscopy. From the study of conserved variants its origin was determined to be Y167 which is surprising as this residue is not part of the active site. Upon inspection of the active site it becomes evident that W272 could be the actual donor of the missing electron, which can then be replenished from Y167 or from the Y280-H276 cross link in the natural cycle. To address the question, whether such a direct electron transfer pathway to the binuclear centre exists two tryptophan 272 variants in subunit I have been generated. These variants are characterised by their turnover rates as well as using EPR and optical spectroscopy. From these experiments it is concluded, that W272 is an important intermediate in the formation of the radical species appearing in P(M) and F* intermediates produced with hydrogen peroxide. The significance of this finding for the catalytic function of the enzyme is discussed.     

Effect of hydrogen peroxide in redox status estimation using nitroxyl spin probe

A procedure for estimating in vivo redox status using EPR and a hydrogen peroxide (H₂O₂)-dependent spin probe method is described. The mechanism of decreasing spin clearance in the selenium-deficient (SeD) rat is discussed. The in vivo decay constant of the nitroxyl spin probe in the liver region of SeD rats appeared to be slightly lower that of the selenium-adequate control (SeC) group, and was significantly smaller than that of normal rats. Bile H₂O₂ levels in normal rats were significantly lower than those in SeD rats. The in vivo decay constant of the spin probe in SeD rats depended on the bile H₂O₂ level. Furthermore, H₂O₂ was detected in the bile in all SeD rats, whereas bile H₂O₂ could be detected in only half of the normal rats. It was found that the in vivo decay constant of the spin probe in normal rats also depended on whether bile H₂O₂ was detected or not. In vivo decay constants were smaller in rats subjected to the surgical operation than in the nonoperated groups. The EPR signal of the nitroxyl radical in the liver homogenate was increased by addition of H₂O₂, which was administered 30 min before the rat was killed. It appears that H₂O₂ can oxidize the hydroxylamine formed following reduction of the spin probe in the liver.     

Kinetic evidence for the re-definition of electron transfer pathways from cytochrome c to O2 within cytochrome oxidase

The reaction with O2 of equimolar mixtures of cytochrome c and cytochrome c oxidase in high and low ionic strength buffers has been examined by flow-flash spectrophotometry at room temperature. In low ionic strength media where cytochrome c and the oxidase are bound in an electrostatic, 1:1 complex some of the cytochrome c is oxidised at a faster rate than a metal centre of the oxidase. In contrast, when cytochrome c and cytochrome c oxidase are predominantly dissociated at high ionic strength cytochrome c oxidation occurs only slowly (t1/2 = 5 s) following the complete oxidation of the oxidase. These results demonstrate that maximal rates of electron transfer from cytochrome c to O2 occur when both substrates are present on the enzyme. The heterogeneous oxidation of cytochrome c observed in the complex implies more than one route for electron transfer within the enzyme. Possibilities for new electron transfer pathways from cytochrome c to O2 are proposed.     

Isolated perfused rat hearts release secondary free radicals during ischemia reperfusion injury: Cardiovascular effects of the spin trap α-phenyl N-tert-butylnitrone

Free radicals produced during myocardial post-ischemic reperfusion are aggravating factors for functional disturbances and cellular injury. The aim of our work was to investigate the significance of the secondary free radical release during non ischemic perfusion and post-ischemic reperfusion and to evaluate the cardiovascular effects of the spin trap used. For that purpose, isolated perfused rat hearts underwent 0, 20, 30 or 60 min of a total ischemia, followed by 30 min of reperfusion. The spin trap: α-phenyl N-tert-butylnitrone (PBN) was used (3 mM). Functional parameters were recorded and samples of coronary effluents were collected and analyzed using Electron Paramagnetic Resonance (EPR) to identify and quantify the amount of spin adducts produced. During non ischemic perfusion, almost undetectable levels of free radical release were observed. Conversely, a large and long-lasting (30 min) release of spin adducts was detected from the onset of reperfusion. The free radical species were identified as alkyl and alkoxyl radicals with amounts reaching 40 times the pre-ischemic values. On the other hand, PBN showed a cardioprotective effect, allowing a significant reduction of rhythm disturbances and a better post-ischemic recovery for the hearts which were submitted to 20 min of ischemia. When the duration of ischemia increased, the protective effects of PBN disappeared and toxic effects became more important. Our results have therefore confirmed the antioxidant and protective properties of a spin trap agent such as PBN. Moreover, we demonstrated that the persistent post-ischemic dysfunction was associated with a sustained production and release of free radical species.     

A comparative study of EPR spin trapping and cytochrome c reduction techniques for the measurement of superoxide anions

Superoxide anions (O₂^.−) generated by the reaction of xanthine with xanthine oxidase were measured by the reduction of cytochrome c and by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the relative sensitivities of these two techniques for the measurement of O₂^.−. Mixtures of xanthine, xanthine oxidase, DMPO generated two adducts, a transient DMPO-OOH and a smaller but longer-lived DMPO-OH. Both adducts were inhibited by superoxide dismutase (SOD), demonstrating they originated from O₂^.−, and were also significantly decreased when the experiments were performed using unchelated buffers, suggesting that metal ion impurities in unchelated buffers alter the formation or degradation of DMPO-adducts. O₂^.−, generated by concentrations of xanthine as low as 0.05 μM, were detectable using EPR spin trapping. In contrast, mixtures of xanthine, xanthine oxidase, and cytochrome c measured spectrophotometrically at 550 nm demonstrated that concentrations of xanthine above 1 μM were required to produce measurable levels of reduced cytochrome c. These studies demonstrate that spin trapping using DMPO was at least 20-fold more sensitive than the reduction of cytochrome c for the measurement of superoxide anions. However, at levels of superoxide generation where cytochrome c provides a linear measurement of production, EPR spin trapping may underestimate radical production, probably due to degradation of DMPO radical adducts.     

Mitochondrial proteome: cancer-altered metabolism associated with cytochrome c oxidase subunit level variation

Shifts in metabolism associated with tumorigenesis were first noted by Otto Warburg in the 1920s. In the ensuing decades many examples of the phenomenon have been elucidated while the underlying molecular mechanism has remained elusive. As the enzyme complex at the crux of oxidative phosphorylation, cytochrome c oxidase is uniquely positioned to have a very high impact on cellular metabolism. In this study, we test the hypothesis that there is a specific association between altered cytochrome c oxidase subunit levels and altered metabolism by combining the technique of reverse-phase protein microarray with radiolabeled glucose metabolic studies. Such a relationship is observed with five different cell lines, two of which (1542N and 1542T) are a matched set of normal and tumor-based lineages derived from the same prostate gland. By measuring the [(14)C]carbon dioxide production of a cell line metabolizing [1-(14)C]glucose and comparing those measurements to values obtained for the same cell line metabolizing [6-(14)C]glucose, we determined the relative utilization of the hexose monophosphate shunt and glycolysis progressing through the Krebs cycle metabolic pathway in each cell line. In all cases there is an increased utilization of hexose monophosphate shunt relative to glycolysis progressing through the Krebs cycle in tumor derived relative to normal derived cell lines. Additionally, there is an associated increase in the ratio of nuclear encoded cytochrome c oxidase subunits to mitochondrially encoded cytochrome c oxidase subunits in the tumor-derived cell lines. These results demonstrate an alteration in subunit levels of a single enzyme complex (cytochrome c oxidase) commensurate with tumor-altered metabolism.     

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.     

Formation of artifactual DMPO-OH spin adduct in acid solutions containing nitrite ions

We investigated aqueous solutions containing nitrite ions and DMPO (5,5-dimethyl-1-pyrroline-N-oxide) by electron spin resonance (ESR) in the pH range from 1 to 6. A DMPO-OH signal was observed below pH 3.0 in the presence of nitrite ions, whereas in the absence of nitrite ion, an extremely weak signal was observed below pH 1.5. Addition of methanol, a hydroxyl radical scavenger, to this system did not lead to the appearance of a detectable DMPO-CH₂OH signal. The possibility of this DMPO-OH signal being due to a genuine spin trapping process with hydroxyl radical was, therefore, ruled out. The reactivities of reactive nitrogen species (RNS) in this system with DMPO have also been investigated by density functional theory (DFT) at the IEFPCM (water)/B3LYP/6–311?+?G ** level of theory. On the basis of the pH dependence of the signal intensity and the redox potential E° (versus SHE) calculated by DFT theory, we propose that the origin of this signal is “inverted spin trapping” via one-electron oxidation of DMPO by H₂ONO⁺, followed by the nucleophilic addition of water. Prevention of these false-positive results when detecting hydroxyl radical using ESR spin trapping requires an awareness of both the presence of nitrite ions in the solution and the solution pH.     

Spin trapping combined with quantitative mass spectrometry defines free radical redistribution within the oxidized hemoglobin:haptoglobin complex

Extracellular or free hemoglobin (Hb) accumulates during hemolysis, tissue damage, and inflammation. Heme-triggered oxidative reactions can lead to diverse structural modifications of lipids and proteins, which contribute to the propagation of tissue damage. One important target of Hb׳s peroxidase reactivity is its own globin structure. Amino acid oxidation and crosslinking events destabilize the protein and ultimately cause accumulation of proinflammatory and cytotoxic Hb degradation products. The Hb scavenger haptoglobin (Hp) attenuates oxidation-induced Hb degradation. In this study we show that in the presence of hydrogen peroxide (H₂O₂), Hb and the Hb:Hp complex share comparable peroxidative reactivity and free radical generation. While oxidation of both free Hb and Hb:Hp complex generates a common tyrosine-based free radical, the spin-trapping reaction with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) yields dissimilar paramagnetic products in Hb and Hb:Hp, suggesting that radicals are differently redistributed within the complex before reacting with the spin trap. With LC-MS² mass spectrometry we assigned multiple known and novel DMPO adduct sites. Quantification of these adducts suggested that the Hb:Hp complex formation causes extensive delocalization of accessible free radicals with drastic reduction of the major tryptophan and cysteine modifications in the β-globin chain of the Hb:Hp complex, including decreased βCys93 DMPO adduction. In contrast, the quantitative changes in DMPO adduct formation on Hb:Hp complex formation were less pronounced in the Hb α-globin chain. In contrast to earlier speculations, we found no evidence that free Hb radicals are delocalized to the Hp chain of the complex. The observation that Hb:Hp complex formation alters free radical distribution in Hb may help to better understand the structural basis for Hp as an antioxidant protein.     

Aging, cytochrome oxidase activity, and hydrogen peroxide release by mitochondria

The objective of this study was to explore the possible cause(s) underlying the previously observed, age-related increase in the rate of mitochondrial H₂O₂ release in the housefly. The hypothesis that an imbalance between different respiratory complexes may be a causal factor was tested. Cytochrome c oxidase activity was found to sharply decline in the latter part of the life span of the flies. Effects of different substrates and respiratory inhibitors were determined in order to ascertain if a decrease in cytochrome c oxidase activity could be responsible for the increased H₂O₂ release. H₂O₂ was measured spectrofluorometrically using horseradish peroxidase and p-hydrophenylacetate as an indicator. Neither NADH-linked substrates nor succinate caused a stimulation of H₂O₂ production. H₂O₂ release by mitochondria, inhibited with rotenone and antimycin A, was greatly increased upon supplementation with -glycerophosphate; however, the further addition of KCN or myxothiazol, to such preparations, caused a depression of H₂O₂ generation. In contrast, relatively low concentrations of KCN or myxothiazol were found to stimulate H₂O₂ release in insect mitochondria supplemented with -glycerophosphate and exposed to rotenone, but not antimycin A. Results are interpreted to suggest that partial inhibition of cytochrome c oxidase activity can lead to the stimulation of mictochondrial H₂O₂ production in the housefly at site(s) other than NADH dehydrogenase and ubisemiquinone/ cytochrome b region; a possible source may be glycerophosphate dehydrogenase.     

A spin label study of conformational changes in cytochrome c

Spin-labeled pig heart cytochromes c singly modified at Met-65, Tyr-74 and at one of the lysine residues, Lys-72 or Lys-73, were investigated by the ESR method under conditions of different ligand and redox states of the heme and at various pH values. Replacement of Met-80 by the external ligand, cyanide, was shown to produce a sharp increase in the mobility of all the three bound labels while reduction of the spin-labeled ferricytochromes c did not cause any marked changes in their ESR spectra. In the pH range 6-13, two conformational transitions in ferricytochrome c were observed which preceded its alkaline denaturation: the first with pK 9.3 registered by the spin label at the Met-65 position, and the second with pK 11.1 registered by the labels bound to Tyr-74 and Lys-72(73). The conformational changes in the 'left-hand part' of ferricytochrome c are most probably induced in both cases by the exchange of internal protein ligands at the sixth coordination site of the heme.     

Identification and quantification of free radicals during myocardial ischemia and reperfusion using electron paramagnetic resonance spectroscopy

There is general agreement that free radicals are involved in reperfusion injury. Electron paramagnetic resonance (EPR) spectroscopy can be considered as the more suitable technique to directly measure and characterize free radical generation during myocardial ischemia and reperfusion. There are essentially two approaches used in the detection of unstable reactive species: freezing technique and spin traps. The detection of secondary free radicals or ascorbyl free radicals during reperfusion might provide an index of oxidative stress. Spin trapping can also characterize nitric oxide. EPR spectroscopy can provide important data regarding redox state and free radical metabolism but ideally, the spin traps must not interfere with cell or organism function.     

Ascorbate peroxidase activity of cytochrome c

Abstract

The peroxidase-type reactivity of cytochrome c is proposed to play a role in free radical production and/or apoptosis. This study describes cytochrome c catalysis of peroxide consumption by ascorbate. Under conditions where the sixth coordination position at the cytochrome c heme iron becomes more accessible for exogenous ligands (by carboxymethylation, cardiolipin addition or by partial denaturation with guanidinium hydrochloride) this peroxidase activity is enhanced. A reaction intermediate is detected by stopped-flow UV-vis spectroscopy upon reaction of guanidine-treated cytochrome c with peroxide, which resembles the spectrum of globin Compound II species and is thus proposed to be a ferryl species. The ability of physiological levels of ascorbate (10–60 µM) to interact with this species may have implications for mechanisms of cell signalling or damage that are based on cytochrome c/peroxide interactions.     

Ataxia telangiectasia mutated influences cytochrome c oxidase activity

Cells lacking ataxia telangiectasia mutated (ATM) have impaired mitochondrial function. Furthermore, mammalian cells lacking ATM have increased levels of reactive oxygen species (ROS) as well as mitochondrial DNA (mtDNA) deletions in the region encoding for cytochrome c oxidase (COX). We hypothesized that ATM specifically influences COX activity in skeletal muscle. COX activity was ∼40% lower in tibialis anterior from ATM-deficient mice than for wild-type mice (P < 0.01, n = 9/group). However, there were no ATM-related differences in activity of succinate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, or complex III. Incubation of wild-type extensor digitorum longus muscles for 1 h with the ATM inhibitor KU55933 caused a ∼50% reduction (P < 0.05, n = 5/group) in COX activity compared to muscles incubated with vehicle alone. Among the control muscles and muscles treated with the ATM inhibitor, COX activity was correlated (r = 0.61, P < 0.05) with activity of glucose 6-phosphate dehydrogenase, a key determinant of antioxidant defense through production of NADPH. Overall, the findings suggest that ATM has a protective role for COX activity.     

The pathomechanism of cytochrome c oxidase deficiency includes nuclear DNA damage

Mitochondrial cytochrome c oxidase (COX, respiratory chain complex IV), contributes to ATP production via oxidative phosphorylation (OXPHOS). Clinical presentation of COX deficiency is heterogeneous ranging from mild to severe neuromuscular diseases. Anemia is among the symptoms and we have previously reported Fanconi anemia like features in COX4-1 deficiency, suggesting genomic instability and our preliminary results detected nuclear double stranded DNA breaks (DSB). We now quantified the DSB by phospho histone H2AX Ser139 staining of COX4-1 and COX6B1 deficient fibroblasts (225% and 215% of normal, respectively) and confirmed their occurrence by neutral comet assay. We further explored the mechanism of DNA damage by studying normal fibroblasts treated with micromolar concentrations of cyanide (KCN). Present results demonstrate elevated nuclear DSB in cells treated with 50?μM KCN for 24?h (170% of normal) in high-glucose medium conditions where ROS and ATP remain normal, although Glutathione content was partially decreased. In glucose-free and serum-free medium, where growth is hampered, DSB were not elevated. Additionally we demonstrate the benefit of nicotinamide riboside (NR) which ameliorated DSB in COX4-1, COX6B1 and KCN treated cells (130%, 154% and 87% of normal cells, respectively). Conversely a negative effect of a poly[ADP-ribose] polymerase (PARP) inhibitor was found. Although additional investigation is needed, our findings raise the possibility that the pathomechanism of COX deficiency and possibly also in other OXPHOS defects, include nuclear DNA damage resulting from nicotinamide adenine dinucleotide (NAD⁺) deficit combined with a replicative state, rather than oxidative stress and energy depletion.     

Designing inhibitors of cytochrome c/cardiolipin peroxidase complexes: mitochondria-targeted imidazole-substituted fatty acids

Mitochondria have emerged as the major regulatory platform responsible for the coordination of numerous metabolic reactions as well as cell death processes, whereby the execution of intrinsic apoptosis includes the production of reactive oxygen species fueling oxidation of cardiolipin (CL) catalyzed by cytochrome (Cyt) c. As this oxidation occurs within the peroxidase complex of Cyt c with CL, the latter represents a promising target for the discovery and design of drugs with antiapoptotic mechanisms of action. In this work, we designed and synthesized a new group of mitochondria-targeted imidazole-substituted analogs of stearic acid TPP-n-ISAs with various positions of the attached imidazole group on the fatty acid (n = 6, 8, 10, 13, and 14). By using a combination of absorption spectroscopy and EPR protocols (continuous wave electron paramagnetic resonance and electron spin echo envelope modulation) we demonstrated that TPP-n-ISAs indeed were able to potently suppress CL-induced structural rearrangements in Cyt c, paving the way to its peroxidase competence. TPP-n-ISA analogs preserved the low-spin hexa-coordinated heme-iron state in Cyt c/CL complexes whereby TPP-6-ISA displayed a significantly more effective preservation pattern than TPP-14-ISA. Elucidation of these intermolecular stabilization mechanisms of Cyt c identified TPP-6-ISA as an effective inhibitor of the peroxidase function of Cyt c/CL complexes with a significant antiapoptotic potential realized in mouse embryonic cells exposed to ionizing irradiation. These experimental findings were detailed and supported by all-atom molecular dynamics simulations. Based on the experimental data and computation predictions, we identified TPP-6-ISA as a candidate drug with optimized antiapoptotic potency.     

The use of specific lysine modifications to locate the reaction site of cytochrome c with sulfite oxidase

The reduction of cytochrome c by beef liver sulfite oxidase was found to be strongly inhibited by high ionic strength, indicating the importance of electrostatic interactions to the reaction. The reaction rates of sulfite oxidase with singly trifluoroacetylated or trifluoromethylphenylcarbamylated cytochrome c derivatives were studied to determine the role of individual lysines in the reaction. The reaction rate was decreased by modification of the lysines immediately surrounding the heme crevice, the decreases following the order: Lys 13 > Lys 25 Lys 79 ≈ Lys 87 > Lys 8 ≈ Lys 27 ≈ Lys 72. Modification of lysines 22, 55, 88, 99, and 100 had no effect on the reaction rate. These results indicate that the interaction site on cytochrome c for sulfite oxidase is at the heme crevice region, and overlaps considerable with that for cytochrome oxidase.     

Chromate-mediated free radical generation from cysteine,penicillamine, hydrogen peroxide,and lipid hydroperoxides

The Cr(VI)-mediated free radical generation from cystein, penicillamine, hydrogen peroxide, and model lipid hydroperoxides was investigated utilizing the electron spin resonance (ESR) spin trapping technique. Incubation of Cr(VI) with cysteine (Cys) generated cysteinyl radical. Radical yield depended on the relative concentrations of Cr(VI) and Cys. The radical generation became detectable at a cysteine: Cr(VI) ration of about 5, reached its highest level at a ratio of 30, and declined thereafter. Cr(VI) or Cys alone did not generate a detectable amount of free radicals. Similar results were obtained with penicillamine. Incubation of Cr(VI), Cys or penicillamine adn H₂O₂ led to hydroxyl (·OH) radical generation, which was verified by quantitative competition experiments utilizing ethanol. The mechanism for ·OH radical generation is considered to be a Cr(VI)-mediated Fenton-like reaction. When model lipid hydroperoxides such as t-butylhydroperoxide and cumene hydroperoxide were used in place of H₂O₂, hydroperoxide-derived free radicals were produced. Since thiols, such as Cys, exist in cellular systems at relatively high concentrations, Cr(VI)-mediated free radical generation in the presence of thiols may participate in the mechanisms of Cr(VI)-induced toxicity and carcinogenesis.     

Oxidative lipidomics of apoptosis: redox catalytic interactions of cytochrome c with cardiolipin and phosphatidylserine

The primary life-supporting function of cytochrome c (cyt c) is control of cellular energetic metabolism as a mobile shuttle in the electron transport chain of mitochondria. Recently, cyt c's equally important life-terminating function as a trigger and regulator of apoptosis was identified. This dreadful role is realized through the relocalization of mitochondrial cyt c to the cytoplasm where it interacts with Apaf-1 in forming apoptosomes and mediating caspase-9 activation. Although the presence of heme moiety of cyt c is essential for the latter function, cyt c's redox catalytic features are not required. Lately, two other essential functions of cyt c in apoptosis, that may rely heavily on its redox activity have been suggested. Both functions are directed toward oxidation of two negatively charged phospholipids, cardiolipin (CL) in the mitochondria and phosphatidylserine (PS) in the plasma membrane. In both cases, oxidized phospholipids seem to be essential for the transduction of two distinctive apoptotic signals: one is participation of oxidized CL in the formation of the mitochondrial permeability transition pore that facilitates release of cyt c into the cytosol and the other is the contribution of oxidized PS to the externalization and recognition of PS (and possibly oxidized PS) on the cell surface by specialized receptors of phagocytes. In this review, we present a new concept that cyt c actuates both of these oxidative roles through a uniform mechanism: its specific interactions with each of these phospholipids result in the conversion and activation of cyt c, transforming it from an innocuous electron transporter into a calamitous peroxidase capable of oxidizing the activating phospholipids. We also show that this new concept is compatible with a leading role for reactive oxygen species in the execution of the apoptotic program, with cyt c as the main executioner.