Inhibition of mitochondrial complex IV leads to secondary loss complex II-III activity: implications for the pathogenesis and treatment of mitochondrial encephalomyopathies.

Mitochondrial encephalomyopathies, arising from deficiencies of the electron transport chain (ETC) give rise to a wide clinical spectrum of presentation and are often progressive in nature. The aetiology of mitochondrial encephalomyopathies have yet to be fully elucidated, however, a successive loss of ETC function may contribute to the progressive nature of these disorders. The possibility arises that as a consequence of a primary impairment of ETC activity, secondary damage to the ETC may occur. In order to investigate this hypothesis, we established a model of cytochrome oxidase (Complex IV) deficiency in cultured human astrocytoma 1321N cells. Potassium cyanide (KCN, 1mM) resulted in a sustained 50% (p<0.01) loss of complex IV. At 24h activities of the other ETC complexes were unaffected. However, at 72h significant loss of succinate-cytochrome c reductase (complex II-III) activity expressed as a ratio to the mitochondrial marker, citrate synthase was observed. (KCN treated; 0.065+/-0.011 vs controls; 0.118+/-0.017 mean+/-SEM, n=8, p<0.05). These results provide a possible mechanism for the progressive nature of ETC defects and why in some patients multiple patterns of ETC deficiencies can be demonstrated.     

Regulation of mitochondrial respiration and apoptosis through cell signaling: cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation

Cytochrome c (Cytc) and cytochrome c oxidase (COX) catalyze the terminal reaction of the mitochondrial electron transport chain (ETC), the reduction of oxygen to water. This irreversible step is highly regulated, as indicated by the presence of tissue-specific and developmentally expressed isoforms, allosteric regulation, and reversible phosphorylations, which are found in both Cytc and COX. The crucial role of the ETC in health and disease is obvious since it, together with ATP synthase, provides the vast majority of cellular energy, which drives all cellular processes. However, under conditions of stress, the ETC generates reactive oxygen species (ROS), which cause cell damage and trigger death processes. We here discuss current knowledge of the regulation of Cytc and COX with a focus on cell signaling pathways, including cAMP/protein kinase A and tyrosine kinase signaling. Based on the crystal structures we highlight all identified phosphorylation sites on Cytc and COX, and we present a new phosphorylation site, Ser126 on COX subunit II. We conclude with a model that links cell signaling with the phosphorylation state of Cytc and COX. This in turn regulates their enzymatic activities, the mitochondrial membrane potential, and the production of ATP and ROS. Our model is discussed through two distinct human pathologies, acute inflammation as seen in sepsis, where phosphorylation leads to strong COX inhibition followed by energy depletion, and ischemia/reperfusion injury, where hyperactive ETC complexes generate pathologically high mitochondrial membrane potentials, leading to excessive ROS production. Although operating at opposite poles of the ETC activity spectrum, both conditions can lead to cell death through energy deprivation or ROS-triggered apoptosis.     

Identification of the binding site on cytochrome c1 for cytochrome c

The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.     

Small scale preparation of skeletal muscle mitochondria, criteria of integrity, and assays with reference to tissue function

Mitochondria prepared in small scale from skeletal muscle were studied with respiration measurements and low temperature spectroscopy. The method of preparation was developed for 25–100 mg tissue with pigeon breast muscle as model organ. The yield was 40%. Data collected during the developmental work were used to evaluate criteria of mitochondrial quality. The cytochrome c conservation, i.e. cytochrome c per mitochondrial quantity in the preparation relative to that in the tissue, is a most useful test parameter. It is bounded between 0–100%. Proportionality between the state 3 rate and the cytochrome c conservation was not rejected by statistical tests. The respiratory control ratio (RCR) was also highly correlated to the cytochrome c conservation. These correlations might be extrapolated to 100% conservation to give hypothetical tissue values. The cause for the correlations is discussed. The P/O ratio showed only weak dependence on the cytochrome c conservation and the state 4 rate s howed no dependence. Other, rather insensitive test parameters are also discussed. The pigeon breast muscle mitochondria isolated by the final method showed cytochrome c conservation of 73 ± 9% (n = 16). They are compared with pig biceps femoris mitochondria prepared by the same method. The two types of mitochondria show many similarities. Some differences may be explained by a different amount of inner mitochondrial membrane relative to mitochondrial protein. The pig tissue contains ten times less mitochondrial protein than the pigeon tissue does. (Mol Cell Biochem 174: 55–60, 1997)     

Measuring the functionality of the mitochondrial pumping complexes with multi-wavelength spectroscopy

The proton pumps of the mitochondrial electron transport chain (ETC) convert redox energy into the proton motive force (ΔP), which is subsequently used by the ATP synthase to regenerate ATP. The limited available redox free energy requires the proton pumps to operate close to equilibrium in order to maintain a high ΔP, which in turn is needed to maintain a high phosphorylation potential. Current biochemical assays measure complex activities far from equilibrium and so shed little light on their function under physiological conditions. Here we combine absorption spectroscopy of the ETC hemes, NADH fluorescence spectroscopy and oxygen consumption to simultaneously measure the redox potential of the intermediate redox pools, the components of ΔP and the electron flux in RAW 264.7 mouse macrophages. We confirm that complex I and III operate near equilibrium and quantify the linear relationship between flux and disequilibrium as a metric of their function under physiological conditions. In addition, we quantify the dependence of complex IV turnover on ΔP and the redox potential of cytochrome c to determine the complex IV driving force and find that the turnover is proportional to this driving force. This form of quantification is a more relevant metric of ETC function than standard biochemical assays and can be used to study the effect of mutations in either mitochondrial or nuclear genome affecting mitochondrial function, post-translation changes, different subunit isoforms, as well as the effect of pharmaceuticals on ETC function.     

Interaction of cytochrome c with cytochrome c oxidase studied by monoclonal antibodies and a protein modifying reagent.

C/57 black mice were immunized with beef heart cytochrome c oxidase, generating 48 hybrid cell lines that secrete antibodies against the different subunits of the enzyme. Immunoblot analysis showed reactions with 7 of the 13 subunits. Among the monoclonal antibodies produced, only those to subunit II gave significant inhibition; these inhibited the enzyme activity completely and prevented cytochrome c binding to the enzyme. Epitope mapping studies indicate that a peptide including residues 200-227 reacts with the antibody, suggesting that the C-terminus of the protein is essential for the binding of this antibody. The carboxyl modifying reagent 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) was chosen to investigate further the relationship between antibody and cytochrome c binding domains. ETC caused 50% inhibition of the enzyme activity with a first-order time during the first 20 min; a slower reaction over 3 h resulted in 90% inhibition. Cytochrome c binding to the oxidase was inhibited to a similar extent as cytochrome c oxidation, and protection against both effects was afforded by the presence of cytochrome c during ETC modification. Anion-exchange of FPLC of the modified forms of cytochrome oxidase revealed extensive inhomogeneity, indicating random derivatization of a number of different carboxyls even during the first-order reaction, and precluding identification of carboxyl residues related to a specific phase of the reaction. Cytochrome c and the subunit II-specific antibody protected against radioactive labeling of subunit II by ETC in the presence of [14C]glycine ethyl ester, demonstrating that the antibody and cytochrome c occupy significant and overlapping areas on the subunit II surface.(ABSTRACT TRUNCATED AT 250 WORDS)     

Initiation of Electron Transport Chain Activity in the Embryonic Heart Coincides with the Activation of Mitochondrial Complex 1 and the Formation of Supercomplexes

Mitochondria provide energy in form of ATP in eukaryotic cells. However, it is not known when, during embryonic cardiac development, mitochondria become able to fulfill this function. To assess this, we measured mitochondrial oxygen consumption and the activity of the complexes (Cx) 1 and 2 of the electron transport chain (ETC) and used immunoprecipitation to follow the generation of mitochondrial supercomplexes. We show that in the heart of mouse embryos at embryonic day (E) 9.5, mitochondrial ETC activity and oxidative phosphorylation (OXPHOS) are not coupled, even though the complexes are present. We show that Cx-1 of the ETC is able to accept electrons from the Krebs cycle, but enzyme assays that specifically measure electron flow to ubiquinone or Cx-3 show no activity at this early embryonic stage. At E11.5, mitochondria appear functionally more mature; ETC activity and OXPHOS are coupled and respond to ETC inhibitors. In addition, the assembly of highly efficient respiratory supercomplexes containing Cx-1, -3, and -4, ubiquinone, and cytochrome c begins at E11.5, the exact time when Cx-1 becomes functional activated. At E13.5, ETC activity and OXPHOS of embryonic heart mitochondria are indistinguishable from adult mitochondria. In summary, our data suggest that between E9.5 and E11.5 dramatic changes occur in the mitochondria of the embryonic heart, which result in an increase in OXPHOS due to the activation of complex 1 and the formation of supercomplexes.     

The impact of the thermal sensitivity of cytochrome c oxidase on the respiration rate of Arctic charr red muscle mitochondria

To assess if cytochrome c oxidase could determine the response of mitochondrial respiration to changes in environmental temperature in ectotherms, we performed KCN titration of the respiration rate and cytochrome c oxidase activity in mitochondria from Arctic charr (Salvelinusfontinalis) muscle at four different temperatures (1 degrees C, 6 degrees C, 12 degrees C, and 18 degrees C). Our data showed an excess of cytochrome c oxidase activity over the mitochondrial state 3 respiration rate. Mitochondrial oxygen consumption rates reached approximately 12% of the cytochrome c oxidase maximal capacity at every temperature. Also, following titration, the mitochondrial respiration rate significantly decreased when KCN reached concentrations that inhibit almost 90% of the cytochrome c oxidase activity. This strongly supports the idea that the thermal sensitivity of the maximal mitochondrial respiration rate cannot be dictated by the effect of temperature on cytochrome c oxidase catalytic capacity. Furthermore, the strong similarity of the Q10s of mitochondrial respiration and cytochrome c oxidase activity suggests a functional or structural link between the two. The functional link could be coevolution of parts of the mitochondrial system to maintain optimal functions in most of the temperature range encountered by organisms.     

Accelerated evolution of the electron transport chain in anthropoid primates

Mitochondria are both the power plant of the cell and a central integrator of signals that govern the lifespan, replication and death of the cell. Perhaps as a consequence, genes that encode components of the mitochondrial electron transport chain (ETC) are generally conserved. Therefore, it is surprising that many of these genes in anthropoid primates (New World monkeys, Old World monkeys and apes, including humans) have been major targets of darwinian positive selection. Sequence comparisons have provided evidence that marked increases of non-synonymous substitution rates occurred in anthropoid ETC genes that encode subunits of Complex III and IV, and the electron carrier molecule cytochrome c (CYC). Two important questions are: (i) how has evolution altered ETC function? and; (ii) how might functional changes in the ETC be linked to evolution of an expanded neocortical brain?     

Decline in cytochrome c oxidase activity in rat-brain mitochondria with aging. Role of peroxidized cardiolipin and beneficial effect of melatonin

Reactive oxygen species (ROS) are considered a key factor in mitochondrial dysfunction associated with brain aging process. Mitochondrial respiration is an important source of ROS and hence a potential contributor to brain functional changes with aging. In this study, we examined the effect of aging on cytochrome c oxidase activity and other bioenergetic processes such as oxygen consumption, membrane potential and ROS production in rat brain mitochondria. We found a significant age-dependent decline in the cytochrome c oxidase activity which was associated with parallel changes in state 3 respiration, membrane potential and with an increase in H₂O₂ generation. The cytochrome aa₃ content was practically unchanged in mitochondria from young and aged animals. The age-dependent decline of cytochrome c oxidase activity could be restored, in situ, to the level of young animals, by exogenously added cardiolipin. In addition, exposure of brain mitochondria to peroxidized cardiolipin resulted in an inactivation of this enzyme complex. It is suggested that oxidation/depletion of cardiolipin could be responsible, at least in part, for the decline of cytochrome c oxidase and mitochondrial dysfunction in brain aging. Melatonin treatment of old animals largely prevented the age-associated alterations of mitochondrial bioenergetic parameters. These results may prove useful in elucidating the molecular mechanisms underlying mitochondrial dysfunction associated with brain aging process, and may have implications in etiopathology of age-associated neurodegenerative disorders and in the development of potential treatment strategies.     

Activation of Hsp90/NOS and increased NO generation does not impair mitochondrial respiratory chain by competitive binding at cytochrome C Oxidase in low oxygen concentrations

Nitric oxide (NO) is known to regulate mitochondrial respiration, especially during metabolic stress and disease, by nitrosation of the mitochondrial electron transport chain (ETC) complexes (irreversible) and by a competitive binding at O₂ binding site of cytochrome c oxidase (CcO) in complex IV (reversible). In this study, by using bovine aortic endothelial cells, we demonstrate that the inhibitory effect of endogenously generated NO by nitric oxide synthase (NOS) activation, by either NOS stimulators or association with heat shock protein 90 (Hsp90), is significant only at high prevailing pO₂ through nitrosation of mitochondrial ETC complexes, but it does not inhibit the respiration by competitive binding at CcO at very low pO₂. ETC complexes activity measurements confirmed that significant reduction in complex IV activity was noticed at higher pO₂, but it was unaffected at low pO₂ in these cells. This was further extended to heat-shocked cells, where NOS was activated by the induction/activation of (Hsp90) through heat shock at an elevated temperature of 42°C. From these results, we conclude that the entire attenuation of respiration by endogenous NO is due to irreversible inhibition by nitrosation of ETC complexes but not through reversible inhibition by competing with O₂ binding at CcO at complex IV.     

Mammalian liver cytochrome c is tyrosine-48 phosphorylated in vivo, inhibiting mitochondrial respiration

Cytochrome c (Cyt c) is part of the mitochondrial electron transport chain (ETC), accepting electrons from bc(1) complex and transferring them to cytochrome c oxidase (CcO). The ETC generates the mitochondrial membrane potential, which is used by ATP synthase to produce ATP. In addition, the release of Cyt c from the mitochondria often commits a cell to undergo apoptosis. Considering its central role in life (respiration) and death (apoptosis) decisions one would expect tight regulation of Cyt c function. Reversible phosphorylation is a main cellular regulatory mechanism, but the effect of cell signaling targeting the mitochondrial oxidative phosphorylation system is not well understood, and only a small number of proteins that can be phosphorylated have been identified to date. We have recently shown that Cyt c isolated from cow heart tissue is phosphorylated on tyrosine 97 in vivo, which leads to inhibition of respiration in the reaction with CcO. In this study we isolated Cyt c from a different organ, cow liver, under conditions preserving the physiological phosphorylation state. Western analysis with a phosphotyrosine specific antibody suggested that liver Cyt c is phosphorylated. Surprisingly, the phosphorylation site was unambiguously assigned to Tyr-48 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS), and not to the previously identified phospho-Tyr-97 in cow heart. As is true of Tyr-97, Tyr-48 is conserved in eukaryotes. As one possible consequence of Tyr-48 phosphorylation we analyzed the in vitro reaction kinetics with isolated cow liver CcO revealing striking differences. Maximal turnover of Tyr-48 phosphorylated Cyt c was 3.7 s(-1) whereas dephosphorylation resulted in a 2.2 fold increase in activity to 8.2 s(-1). Effects of Tyr-48 phosphorylation based on the Cyt c crystal structure are discussed.     

Effects of weight loss and physical activity on skeletal muscle mitochondrial function in obesity

The current study was undertaken to address responsiveness of skeletal muscle mitochondrial electron transport chain (ETC) activity to weight loss (WL) and exercise in overweight or obese, sedentary volunteers. Fourteen middle-aged participants (7 male/7 female) had assessments of mitochondrial ETC activity and mitochondrial (mt)DNA in vastus lateralis muscle, obtained by percutaneous biopsy, before and after a 16-wk intervention. Mean WL was 9.7 (1.5%) and the mean increase in Vo(2 max) was [means (SD)] 21.7 (3.7)%. Total ETC activity increased significantly, from 0.13 (0.02) to 0.19 (0.03) U/mU creatine kinase (CK; P < 0.001). ETC activity was also assessed in mitochondria isolated into subsarcolemmal (SSM) and intermyofibrillar (IMF-M) fractions. In response to intervention, there was a robust increase of ETC activity in SSM (0.028 (0.007) to 0.046 (0.011) U/mU CK, P < 0.001), and in IMF-M [0.101 (0.015) to 0.148 (0.018) U/mU CK, P < 0.005]. At baseline, the percentage of ETC activity contained in the SSM fraction was low and remained unchanged following intervention [19 (3) vs. 22 (2)%], despite the increase in ETC activity. Also, muscle mtDNA content did not change significantly [1665 (213) vs. 1874 (214) mtDNA/nuclear DNA], denoting functional improvement rather than proliferation of mitochondria as the principal mechanism of enhanced ETC activity. Increases in ETC activity were correlated with energy expenditure during exercise sessions, and ETC activity in SSM correlated with insulin sensitivity after adjustment for Vo(2 max). In summary, skeletal muscle ETC activity is increased by WL and exercise in previously sedentary obese men and women. We conclude that improved skeletal muscle ETC activity following moderate WL and improved aerobic capacity contributes to associated alleviation of insulin resistance.     

Impairment of mitochondrial respiratory chain activity in aortic endothelial cells induced by glycated low-density lipoprotein

Coronary artery disease (CAD) is the leading cause of mortality in diabetic patients. Mitochondrial dysfunction and increased production of reactive oxygen species (ROS) are associated with diabetes and CAD. Elevated levels of glycated LDL (glyLDL) were detected in patients with diabetes. Our previous studies demonstrated that glyLDL increased the generation of ROS and altered the activities of antioxidant enzymes in vascular endothelial cells (EC). This study examined the effects of glyLDL on oxygen consumption in mitochondria and the activities of key enzymes in the mitochondrial electron transport chain (ETC) in cultured porcine aortic EC. The results demonstrated that glyLDL treatment significantly impaired oxygen consumption in Complexes I, II/III, and IV of the mitochondrial ETC in EC compared to LDL or vehicle control detected using oxygraphy. Incubation with glyLDL significantly reduced the mitochondrial membrane potential, the NAD⁺/NADH ratio, and the activities of mitochondrial ETC enzymes (NADH-ubiquinone dehydrogenase, succinate cytochrome c reductase, ubiquinone cytochrome c reductase, and cytochrome c oxidase) in EC compared to LDL or control. The abundance of mitochondria-associated ROS and the release of ROS from EC were significantly increased after glyLDL treatment. The findings suggest that glyLDL attenuates the activities of key enzymes in the mitochondrial ETC, decreases mitochondrial oxygen consumption, reduces mitochondrial membrane potential, and increases ROS generation in EC, which potentially contribute to mitochondrial dysfunction in diabetic patients.     

Electron transport chain in a thermotolerant yeast

Yeasts capable of growing and surviving at high temperatures are regarded as thermotolerant. For appropriate functioning of cellular processes and cell survival, the maintenance of an optimal redox state is critical of reducing and oxidizing species. We studied mitochondrial functions of the thermotolerant Kluyveromyces marxianus SLP1 and the mesophilic OFF1 yeasts, through the evaluation of its mitochondrial membrane potential (ΔΨ_m), ATPase activity, electron transport chain (ETC) activities, alternative oxidase activity, lipid peroxidation. Mitochondrial membrane potential and the cytoplasmic free Ca²⁺ ions (Ca²⁺ cyt) increased in the SLP1 yeast when exposed to high temperature, compared with the mesophilic yeast OFF1. ATPase activity in the mesophilic yeast diminished 80% when exposed to 40° while the thermotolerant SLP1 showed no change, despite an increase in the mitochondrial lipid peroxidation. The SLP1 thermotolerant yeast exposed to high temperature showed a diminution of 33% of the oxygen consumption in state 4. The uncoupled state 3 of oxygen consumption did not change in the mesophilic yeast when it had an increase of temperature, whereas in the thermotolerant SLP1 yeast resulted in an increase of 2.5 times when yeast were grown at 30^o, while a decrease of 51% was observed when it was exposed to high temperature. The activities of the ETC complexes were diminished in the SLP1 when exposed to high temperature, but also it was distinguished an alternative oxidase activity. Our results suggest that the mitochondria state, particularly ETC state, is an important characteristic of the thermotolerance of the SLP1 yeast strain.     

Quantitative proteomic analysis of mitochondrial proteins reveals prosurvival mechanisms in the perpetuation of radiation-induced genomic instability

Radiation-induced genomic instability is a well-studied phenomenon that is measured as mitotically heritable genetic alterations observed in the progeny of an irradiated cell. The mechanisms that perpetuate this instability are unclear; however, a role for chronic oxidative stress has consistently been demonstrated. In the chromosomally unstable LS12 cell line, oxidative stress and genomic instability were correlated with mitochondrial dysfunction. To clarify this mitochondrial dysfunction and gain insight into the mechanisms underlying radiation-induced genomic instability we have evaluated the mitochondrial subproteome and performed quantitative mass spectrometry analysis of LS12 cells. Of 98 quantified mitochondrial proteins, 17 met criteria for fold changes and reproducibility; and 11 were statistically significant in comparison with the stable parental GM10115 cell line. Previous observations implicated defects in the electron transport chain (ETC) in the LS12 cell mitochondrial dysfunction. Proteomic analysis supports these observations, demonstrating significantly reduced levels of mitochondrial cytochrome c, the intermediary between complexes III and IV of the ETC. Results also suggest that LS12 cells compensate for ETC dysfunction and oxidative stress through increased levels of tricarboxylic acid cycle enzymes and upregulation of proteins that protect against oxidative stress and apoptosis. More than one cellular defect is likely to contribute to the genomic instability phenotype, and evaluation of gene and microRNA expression suggests that epigenetics play a role in the phenotype. These data suggest that LS12 cells have adapted mechanisms that allow survival under suboptimal conditions of oxidative stress and compromised mitochondrial function to perpetuate genomic instability.     

Kinetic alterations of cytochrome c oxidase in carbon tetrachloride induced cirrhotic rat liver

In a study of the chronic effects of CCl4 on the respiratory activities of rat liver mitochondria, the content of cytochrome c oxidase increased from 0.077 +/- 0.010 (nmol/mg protein) for normal rats to 0.101 +/- 0.009, and its specific activity increased from Vmax = 345 +/- 24 (e-/s/cytochrome aa3) to 431 +/- 19 in mitochondria of CCl4 treated rats. There was a slight increase in Km for cytochrome c from 5.63 +/- 0.08 microM to 7.79 +/- 0.80. These results would strongly suggest that an appreciable decrease in the steady state concentration of ATP in hepatic cells of CCl4 treated rats brought about a compensatory increase in the overall activity of cytochrome c oxidase. However, when the rate of oxygen uptake by mitochondria was measured in the presence of rotenone and tetramethyl-p-phenylene-diamine with NADH as substrate, the specific activity in CCl4 treated rats was lower than that of normal rats (Vmax = 345 +/- 31 (e-/s/cytochrome aa3), as compared to Vmax = 408 +/- 21) in spite of the increased activity of cytochrome c oxidase. This phenomenon was ascribed to a decrease in the activity of NADH cytochrome b5 reductase in the mitochondrial outer membrane due to CCl4 treatment.     

Resonance Raman evidence for low-spin Fe2+ heme a3 in energized cytochrome c oxidase: implications for the inhibition of O2 reduction

Resonance Raman (RR) spectra are reported for reduced submitochondrial particles (SMP) with excitation at 441.6 nm, where Raman bands of the cytochrome c oxidase heme a groups are selectively enhanced. Addition of ATP to energize the membranes induces the formation of a new band at 1644 cm-1 and partial loss of intensity in a band at 1567 cm-1. These changes are modeled by adding cyanide to reduced cytochrome c oxidase and are attributed to partial conversion of cytochrome (cyt) a3 from a high-spin to a low-spin state. This conversion is abolished by addition of excess oligomycin, an ATPase inhibitor, or FCCP, an uncoupler of proton translocation, and is reversed when the ATP is consumed. The observed spin-state conversion is attributed to the binding of an endogenous ligand to the cyt a3 Fe atom. This ligation is suggested to be induced by a local increase in pH and/or by a global conformation change associated with the generation of a transmembrane potential. Since O2 binding requires a vacant coordination site at cyt a3, the ligation of this site must retard O2 reduction and could thus provide a simple mechanism for energy-linked regulation of respiration. No changes in the RR spectrum were observed upon adding Ca2+ or H+ to reduced cytochrome c oxidase. The cyt a3 spin-state change associated with membrane energization is unrelated to the cyt a absorption red shift induced by adding Ca2+ or H+ to cytochrome c oxidase.     

Complex I-Associated Hydrogen Peroxide Production Is Decreased and Electron Transport Chain Enzyme Activities Are Altered in n-3 Enriched fat-1 Mice

The polyunsaturated nature of n-3 fatty acids makes them prone to oxidative damage. However, it is not clear if n-3 fatty acids are simply a passive site for oxidative attack or if they also modulate mitochondrial reactive oxygen species (ROS) production. The present study used fat-1 transgenic mice, that are capable of synthesizing n-3 fatty acids, to investigate the influence of increases in n-3 fatty acids and resultant decreases in the n-6∶n-3 ratio on liver mitochondrial H₂O₂ production and electron transport chain (ETC) activity. There was an increase in n-3 fatty acids and a decrease in the n-6∶n-3 ratio in liver mitochondria from the fat-1 compared to control mice. This change was largely due to alterations in the fatty acid composition of phosphatidylcholine and phosphatidylethanolamine, with only a small percentage of fatty acids in cardiolipin being altered in the fat-1 animals. The lipid changes in the fat-1 mice were associated with a decrease (p<0.05) in the activity of ETC complex I and increases (p<0.05) in the activities of complexes III and IV. Mitochondrial H₂O₂ production with either succinate or succinate/glutamate/malate substrates was also decreased (p<0.05) in the fat-1 mice. This change in H₂O₂ production was due to a decrease in ROS production from ETC complex I in the fat-1 animals. These results indicate that the fatty acid changes in fat-1 liver mitochondria may at least partially oppose oxidative stress by limiting ROS production from ETC complex I.