The possible role of isoforms of cytochrome c oxidase subunit VIa in mammalian thermogenesis

A single cDNA of cytochrome c oxidase subunit VIa was characterised from liver, heart and the thermogenic organ of the partially endotherm tuna fish. The amino acid sequence revealed high identity with subunit VIa from carp and trout, but low identity to subunits VIaL (liver type) and VIaH (heart type) of mammalian cytochrome c oxidase. In reconstituted cytochrome c oxidase from bovine heart, the H ⁺/e⁻ stoichiometry is decreased from 1.0 to 0.5 at high intraliposomal ATP/ADP ratios via exchange of bound ADP by ATP at the matrix domain of the transmembraneous subunit VIaH. Reconstituted cytochrome c oxidase from bovine liver and kidney, containing subunit VIaL, revealed H ⁺/e⁻ ratios below 0.5, independent of the ATP/ADP ratio. The results suggest the evolution of three types of subunit VIa. Subunits VIaH and VIaL are postulated to participate in mammalian thermogenesis. Received 3 May 1999; received after revision 10 June 1999; accepted 29 June 1999     

Control of respiration by nitric oxide in Keilin-Hartree particles,mitochondria and SH-SY5Y neuroblastoma cells

The pattern of cytochrome c oxidase inhibition by nitric oxide (NO) was investigated polarographically using Keilin-Hartree particles, mitochondria and human neuroblastoma cells. NO reacts with purified cytochrome c oxidase forming either a nitrosyl- or a nitrite-inhibited derivative, displaying distinct kinetics and light sensitivity of respiration recovery in the absence of free NO. Keilin-Hartree particles or cells, respiring either on endogenous substrates alone or in the presence of ascorbate, as well as state 3and state 4mitochondria respiring on glutamate and malate, displayed the rapid recovery characteristic of the nitrite derivative. All systems, when respiring in the presence of tetramethyl-p-phenylenediamine, were characterised by the slower, light-sensitive recovery typical of the nitrosyl derivative. Together the results suggest that the reaction of NO with cytochrome c oxidase in situ follows two alternative inhibition pathways, depending on the electron flux through the respiratory chain.Received 1 April 2003; received after revision 22 May 2003; accepted 3 June 2003     

The ratio between the fast and slow forms of bovine cytochrome c oxidase is changed by cholate or nucleotides bound to the cholate-binding site close to the cytochrome a3/CuB binuclear centre

We determined the fraction of 'slow' and 'fast' conformations of bovine cytochrome c oxidase, following the kinetics of cyanide binding to the oxidized enzyme. We investigated whether treatment of heart mitochondrial particles with different commercially available types of cholate (standard and ultrapure) can affect the fraction of cytochrome c oxidase in the two states. Compared to standard cholate, the use of ultra-pure cholate for solubilization of heart mitochondrial particles significantly increased the fraction of the fast enzyme. Complete homogeneity (approximately 100% fast) was observed when cytochrome c oxidase was solubilized with ultra-pure cholate from heart mitochondrial particles pre-equilibrated with AMP; equilibration with ADP yielded a much smaller fraction of fast enzyme (approximately 35%). These observations are discussed on the basis of the structural relationships between the known cholate-binding site and the binuclear cytochrome a3-CuB site: variation in the occupancy of this binding site with cholate or nucleotides may modify reactivity of the oxidized binuclear centre towards cyanide.     

Effects of oral contraceptive steroids (norethisterone/mestranol) on the activities of hepatic drug-metabolizing enzymes in iron-deficient anemic rats

Either oral contraceptive steroid (norethisterone/mestranol; N/M) treatment or iron-deficiency (Fe(-] anemia alone caused an increase in NADPH cytochrome c reductase and in three hepatic microsomal mixed-function oxidase activities in female rats. When N/M treatment and the Fe(-) diet are combined, no further change in hepatic enzyme activity is seen compared with that with either treatment alone.     

Interaction of BW755C,a potent inhibitor of lipoxygenase and cyclo-oxygenase,with mitochondrial cytochrome oxidase

Summary The interaction between BW755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline), a potent inhibitor of both lipoxygenase and cyclo-oxygenase, and respiratory chain in mitochondria and electron transport particles (ETP) from rat livers was examined. BW755C accelerated the oxygen uptake by mitochondria without the addition of substrate for the respiratory chain. Spectrophotometric study revealed that BW755C was quickly oxidized by cytochrome oxidase in mitochondria to a compound possessing an absorption maximum at 524 nm. p-Phenylenediamine (p-diaminobenzene, PPDA), which, like BW755C, serves as an electron donor to cytoschrome oxidase, was shown to inhibit the generation of active oxygen in macrophages; the inhibition was stronger than that of BW755C. These results strongly suggest that the oxidative conversion of BW755C by mitochondrial cytochrome oxidase is associated with its potentially inhibitory action on the active oxygen-generating system in phagocytes.The authors are indebted to Dr M. Hori, Gifu College of Pharmacy and to Dr Y. Orii, Kyoto University for their kind supplies of BW755C and pure cytochrome oxidase, respectively.     

Interaction of BW755C, a potent inhibitor of lipoxygenase and cyclo-oxygenase, with mitochondrial cytochrome oxidase

The interaction between BW755C (3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline), a potent inhibitor of both lipoxygenase and cyclo-oxygenase, and respiratory chain in mitochondria and electron transport particles (ETP) from rat livers was examined. BW755C accelerated the oxygen uptake by mitochondria without the addition of substrate for the respiratory chain. Spectrophotometric study revealed that BW755C was quickly oxidized by cytochrome oxidase in mitochondria to a compound possessing an absorption maximum at 524 nm. p-Phenylenediamine (p-diaminobenzene, PPDA), which, like BW755C, serves as an electron donor to cytochrome oxidase, was shown to inhibit the generation of active oxygen in macrophages; the inhibition was stronger than that of BW755C. These results strongly suggest that the oxidative conversion of BW755C by mitochondrial cytochrome oxidase is associated with its potentially inhibitory action on the active oxygen-generating system in phagocytes.     

Change of the cytochrome oxidase level during exponential growth in Neurospora crassa.

In Neurospora cells growing in various media, the specific activity of cytochrome oxidase increases very markedly during early exponential growth, reaching a maximum after 4-5 duplication times, then it slowly declines.     

Permeability of rat heart myocytes to cytochrome c

Rat heart myocytes undergoing progressive damage demonstrate morphological changes of shortening and swelling followed by the formation of intracellular vacuoles and plasma membrane blebbing. The damaged myocytes displayed impaired N,N'-tetramethyl-p-phenyldiamine (TMPD) ascorbate-stimulated respiratory activity which was restored by the addition of reduced cytochrome c to the cell culture medium. To clarify the role played by cytochrome c in the impairment of cell respiration, polarographic, spectrophotometric and fluorescence as well as electron microscopy imaging experiments were performed. TMPD/ascorbate-stimulated respiratory activity returned to control levels, at approximately 20 microM cytochrome c, establishing the threshold below which the turnover rate by cytochrome c oxidase in the cell depends on cytochrome concentration. Mildly damaged cardiac myocytes, as indicated by cell shortening, retention of visible striations and free-fluorescein exclusion, together with the absence of lactate dehydrogenase leakage and exclusion of trypan blue, were able to oxidize exogenous cytochrome c and were permeable to fluorescein-conjugated cytochrome c. The results, while consistent with an early cytochrome c release observed at the beginning of cell death, elucidate the role played by cytochrome c in the kinetic control of mitochondrial electron transfer under pathological conditions, particularly those involving the terminal part of the respiratory chain. These data are the first to demonstrate that the sarcolemma of cardiac myocytes, damaged but still viable, is permeable to cytochrome c.     

Intermolecular and intramolecular isotope effects in the deamination of putrescine catalyzed by diamine oxidase

The enzymatic deamination of 1,4-diaminobutane (putrescine) catalyzed by hog kidney diamine oxidase was studied with the aid of deuterium labeled substrates and mass spectrometry. An intermolecular deuterium isotope effect for the deamination of putrescine labeled with deuterium in all 4 alpha positions was observed to be 1.26. 1,4-Diaminobutane-1,1-d2 was synthesized and intramolecular isotope effects determined. The preference of diamine oxidase for the unlabeled alpha position was about 4 times greater than for the deuterated methylene. This work shows that intramolecular deuterium isotope effects are observable in enzyme systems other than cytochrome P-450.     

Action de quelques catéchines sur l'activité d'un enzyme (la cytochrome-oxydase) de la chaÎne respiratoire

Summary The effect of 4 different flavonoids on the respiratory activity of cytochrome oxidase in rat liver homogenate was measured in vitro using the Warburg apparatus. The results show that 3 compounds of the catechin group are especially active.     

Morphologische Veränderungen der Extraorbitaldrüsen weisser Ratten nach Kupfermangelernährung

Summary Copper deficiency of the rat leads to a reduction of the histochemically demonstrable cytochrome oxidase activity of the glandula extraorbitalis, and to atrophy and fatty degeneration of the glandular cells. The histological alterations are more pronounced in the male than in the female.     

Ionic control of biochemical reactions]

It is shown that pH and ionic strength are tightly interdependent in cytochrome oxidase activity at the level of inner membranes of the mitochondrion, as a direct consequence of the polyanionic environment of this enzyme. Application of polyelectrolyte theory explains a number of biochemical reactions controlled by ionic strength fluctuations.     

Intermolecular and intramolecular isotope effects in the deamination of putrescine catalyzed by diamine oxidase

Summary The enzymatic deamination of 1,4-diaminobutane (putrescine) catalyzed by hog kidney diamine oxidase was studied with the aid of deuterium labeled substrates and mass spectrometry. An intermolecular deuterium isotope effect for the deamination of putrescine labeled with deuterium in all 4 alpha positions was observed to be 1.26. 1,4-Diaminobutane-1, 1-d₂ was synthesized and intramolecular isotope effects determined. The preference of diamine oxidase for the unlabeled alpha position was about 4 times greater than for the deuterated methylene. This work shows that intramolecular deuterium isotope effects are observable in enzyme systems other than cytochrome P-450.Acknowledgment. This investigation was supported by a research grant from the National Institutes of Health, grant number NS 14017.     

Involvement of reactive oxygen species in the microsomal S-oxidation of thiobenzamide

Superoxide dismutase, catalase and methional proved capable of inhibiting the microsomal oxidation of thiobenzamide, which is most probably catalyzed by the flavin-containing monooxygenase. This indicates that excited oxygen species (e.g. X O-2,H2O2, X OH) are involved in the catalytic cycle of this enzymatic reaction. CO, which inhibits the cytochrome P-450-dependent oxygen radical formation, had no effect on the oxidation reaction, suggesting that the source of the reactive oxygen species is not the microsomal mixed-function oxidase.     

Role of nitric oxide in the functional response to ischemia-reperfusion of heart mitochondria from hyperthyroid rats

We investigated the role of nitric oxide (NO) in the mitochondrial derangement associated with the functional response to ischemia-reperfusion of hyperthyroid rat hearts. Mitochondria were isolated at 3000 g from hearts subjected to ischemia-reperfusion, with or without N-nitro-L-arginine (L-NNA, an NO synthase inhibitor). During reperfusion, hyperthyroid hearts displayed tachycardia and low functional recovery. Their mitochondria exhibited O₂ consumption similar to euthyroid controls, while H₂O₂ production, hydroperoxide, protein-bound carbonyl and nitrotyrosine levels, and susceptibility to swelling were higher. L-NNA blocked the reperfusion tachycardic response and increased inotropic recovery in hyperthyroid hearts. L-NNA decreased mitochondrial H₂O₂ production and oxidative damage, and increased respiration and tolerance to swelling. Such effects were higher in hyperthyroid preparations. These results confirm the role of mitochondria in ischemia-reperfusion damage, and strongly suggest that NO overproduction is involved in the high mitochondrial dysfunction and the low recovery of hyperthyroid hearts from ischemia-reperfusion. L-NNA also decreased protein content and cytochrome oxidase activity of a mitochondrial fraction isolated at 8000 g. This and previous results suggest that the above fraction contains, together with light mitochondria, damaged mitochondria coming from the heaviest fraction, which has the highest cytochrome oxidase activity and capacity to produce H₂O₂. Therefore, we propose that the high mitochondrial susceptibility to swelling, favoring mitochondrial population purification from H₂O₂-overproducing mitochondria, limits hyperthyroid heart oxidative stress.Received 24 March 2004; received after revision 9 June 2004; accepted 5 July 2004     

Involvement of reactive oxygen species in the microsomal S-oxidation of thiobenzamide

Summary Superoxide dismutase, catalase and methional proved capable of inhibiting the microsomal oxidation of thiobenzamide, which is most probably catalyzed by the flavin-containing monooxygenase. This indicates that excited oxygen species (e. g.·O ₂ ^– , H₂O₂, ·OH) are involved in the catalytic cycle of this enzymatic reaction. CO, which inhibits the cytochrome P-450-dependent oxygen radical formation, had no effect on the oxidation reaction, suggesting that the source of the reactive oxygen species is not the microsomal mixed-function oxidase.     

Improving the thermal stability of lactate oxidase by directed evolution

Lactate oxidase is used in biosensors to measure the concentration of lactate in the blood and other body fluids. Increasing the thermostability of lactate oxidase can significantly prolong the lifetime of these biosensors. We have previously obtained a variant of lactate oxidase from Aerococcus viridans with two mutations (E160G/V198I) that is significantly more thermostable than the wild-type enzyme. Here we have attempted to further improve the thermostability of E160G/V198I lactate oxidase using directed evolution. We made a mutant lactate oxidase gene library by applying error-prone PCR and DNA shuffling, and screened for thermostable mutant lactate oxidase using a plate-based assay. After three rounds of screening we obtained a thermostable mutant lactate oxidase, which has six mutations (E160G/V198I/G36S/T103S/A232S/F277Y). The half-life of this lactate oxidase at 70 °C was about 2 times that of E160G/V198I and about 36 times that of the wild-type enzyme. The amino acid mutation process suggests that the combined neutral mutations are important in protein evolution. Received 15 September 2006; received after revision 21 October 2006; accepted 2 November 2006     

香烟烟雾提取物对人内皮细胞细胞色素C氧化酶活性及细胞凋亡的影响

目的探讨香烟烟雾提取物（cigarette smoking extract,CSE）对内皮细胞细胞色素C氧化酶（cytochmme Coxidase,COX）活性及凋亡的影响。方法体外培养ECV304,分别给予0％、0．5％、1％、5％CSE刺激12h,及5％CSE刺激0h、6h、12h、24h后,生化法检测COX活性;投射电镜和流式细胞仪观察细胞凋亡情况。结果CSE引起COX活性下降,且随着刺激浓度和时间的增加而下降（P〈0．05）;电镜示CSE干预组细胞出现明显的凋亡形态学改变;流式细胞仪结果示不同浓度CSE分别作用12h后凋亡率依次增高,除O％CSE组和0．5％CSE组间比较无统计学意义（P〉0．05）,余各组间比较均有统计学意义（P〈0．05）;5％CSE作用不同时间后,随着干预时间的延长细胞凋亡率逐渐升高（P〈0．05）。结论CSE抑制内皮细胞COX活性,呈浓度和时间依赖性;CSE诱导内皮细胞凋亡,呈浓度和时间依赖性;COX活性的下降可能在CSE所致的内皮细胞凋亡中具有重要作用。     

Semiconduction theory

Conclusions The semiconducting properties of biomacromolecules are a certainty which cannot be neglected when models are proposed for explaining some biological function such as, enzymatic activity of cytochrome oxidase, olfactory transduction, visual reception, properties of the cellular membranes, etc.^6,7,9. Although a perfect correlation does not yet exist between the results of molecular orbital calculations and experimental data regarding the electrical conduction in proteins and nucleic acids⁹, a profound study on the aspects connected with intrinsic mechanisms of conduction in biomacromolecules, as well as on dependence of electrical properties on the impurities acting in vivo, should increase our understanding of biophysical phenomena.