Interrelationships of copper, cytochrome oxidase, phospholipid synthesis and adenine nucleotide binding.

Studies are reported on the interrelationships in liver mitochondria of copper status, cytochrome oxidase activity, adenine nucleotide binding capacity and phospholipid synthesis. Direct exposure of mitochondria to cyanide or diethyldithiocarbamate depressed cytochrome oxidase activity; ADP-binding and phospholipid synthesis. Fractionation of mitochondria to increase the specific activity of cytochrome oxidase about 10-fold did not increase the affinity to bind ADP. Ageing of mitochondria or dialysis of mitochondria or mitochondrial membrane preparations against water or diethyldithiocarbamate at 0--2 degrees for 18 h did not decrease cytochrome oxidase activity or copper content of reisolated and resuspended mitochondria or mitochondrial membrane preparations, but considerably reduced the affinity to bind ADP. The respiratory inhibitors, fluoride and azide, at concentrations inhibitory to cytochrome oxidase did not reduce ADP-binding or phospholipid synthesis. Atractyloside did not inhibit cytochrome oxidase activity but did inhibit ADP-binding and phospholipid synthesis. Pre-incubation of mitochondrial membrane preparations with Cu++ increased the copper content and ADP-binding affinity. The results indicate that cytochrome oxidase is not the ADP-binding site of the mitochondrial membrane system and that reduced cytochrome oxidase activity per se does not depress binding affinity. Copper appears to be a component of the adenine nucleotide binding sites of mitochondrial membranes because the copper-complexing agents, cyanide and diethyldithiocarbamate, depressed ADP-binding, while increased mitochondrial membrane copper content increased ADP-binding.     

Regulation of Cytochrome c- and Quinol Oxidases,and Piezotolerance of Their Activities in the Deep-Sea Piezophile Shewanella violacea DSS12 in Response to Growth Conditions

The facultative piezophile Shewanella violacea DSS12 is known to have respiratory components that alter under the influence of hydrostatic pressure during growth, suggesting that its respiratory system is adapted to high pressure. We analyzed the expression of the genes encoding terminal oxidases and some respiratory components of DSS12 under various growth conditions. The expression of some of the genes during growth was regulated by both the O₂ concentration and hydrostatic pressure. Additionally, the activities of cytochrome c oxidase and quinol oxidase of the membrane fraction of DSS12 grown under various conditions were measured under high pressure. The piezotolerance of cytochrome c oxidase activity was dependent on the O₂ concentration during growth, while that of quinol oxidase was influenced by pressure during growth. The activity of quinol oxidase was more piezotolerant than that of cytochrome c oxidase under all growth conditions. Even in the membranes of the non-piezophile Shewanella amazonensis, quinol oxidase was more piezotolerant than cytochrome c oxidase, although both were highly piezosensitive as compared to the activities in DSS12. By phylogenetic analysis, piezophile-specific cytochrome c oxidase, which is also found in the genome of DSS12, was identified in piezophilic Shewanella and related genera. Our observations suggest that DSS12 constitutively expresses piezotolerant respiratory terminal oxidases, and that lower O₂ concentrations and higher hydrostatic pressures induce higher piezotolerance in both types of terminal oxidases. Quinol oxidase might be the dominant terminal oxidase in high-pressure environments, while cytochrome c oxidase might also contribute. These features should contribute to adaptation of DSS12 in deep-sea environments.     

The solid state physical theory of cytochrome oxidase kinetics. Inhibition of second order rate constant,and second to first order kinetic shift with increasing oxygen,predicted from electron injection and trapping

Conduction of electrons through the solid protein cytochrome oxidase particle in accord with Ohm's law, driven by the difference in electrode potentials of two substrates which exchange electrons with the two sides of the enzyme particle, was previously shown to explain the inhibitory effect of cytochromec on the first order rate constant, and to predict the low semiconduction activation energy of dried cytochrome oxidase. If the solid conduction path in the cytochrome oxidase particle shows electron injection from sites of electron exchange with substrate, and shows trapping of conduction electrons by reversible O₂ complexes, then one may also predict that the first order kinetics observed as high O₂ concentrations will change to second order kinetics at lower O₂ concentrations, as observed by Gibson and Wharton. One may also predict quantitatively the inhibitory effect of increasing O₂ concentrations on the second order rate constant as observed by Gibson and Wharton. The same concept of electron trapping by O₂ complexes provides a possible reason for the unusually low semiconduction activation energy of cytochrome oxidase.     

Action of DCCD on the H+/O stoichiometry of mitoplast cytochrome c oxidase

The mechanistic H+/O ejection stoichiometry of the cytochrome c oxidase reaction in rat liver mitoplasts is close to 4 at level flow when the reduced oxidase is pulsed with O2. Dicyclohexylcarbodiimide (DCCD) up to 30 nmol/mg protein fails to influence the rate of electron flow through the mitoplast oxidase, but inhibits H+ ejection. The inhibition of H+ ejection appears to be biphasic; ejection of 2-3 H+ per O is completely inhibited by very low DCCD, whereas inhibition of the remaining H+ ejection requires very much higher concentrations of DCCD. This effect suggests the occurrence of two types of H+ pumps in the native cytochrome oxidase of mitoplasts.     

Fusion of phospholipid vesicles reconstituted with cytochrome c oxidase and mitochondrial hydrophobic protein.

Reconstituted cytochrome oxidase liposomes were fused with liposomes reconstituted with mitochondrial hydrophobic protein, which acts as a membrane-bound uncoupler of cytochrome oxidase. Fusion was assayed by the loss of respiratory control of cytochrome oxidase as measured by the increased rate of ascorbate oxidation induced by hydrophobic protein when both proteins shared the same vesicles. Fusion was dependent on the presence of phosphatidylserine in the liposomes Ca++ in the aqueous medium. Phosphatidylcholine-phosphatidylserine liposomes required higher concentrations of phosphatidylserine and Ca++ than did phosphatidylethanolamine-phosphatidylserine liposomes. Cytochrome oxidase vesicles containing high concentrations of phosphatidylserine showed little or no respiratory control, while those with lower concentrations showed high respiratory control; respiratory control could be induced by fusing cytochrome oxidase vesicles containing high phosphatidylserine with protein-free liposomes containing low phosphatidylserine concentration. If cytochrome oxidase vesicles and hydrophobic protein vesicles were prefused separately for 15 min, they lost the ability to fuse upon being subsequently mixed together. The reconstituted vesicles had diameters of about 200 A; fusion yielded vesicles with diameters in excess of 1000 A.     

The interaction of thioridazine with cardiac cytochrome oxidase; enzyme activity and drug binding studies.

Thioridazine interacts with purified cardiac cytochrome oxidase altering both the activity of the enzyme and the optical spectrum of the drug. Cytochrome oxidase activity, as measured by oxidation of cytochrome c, exhibits a biphasic response to changing drug concentration. Lower concentrations of thioridazine increased cytochrome oxidase activity up to 20% at 65 microM and higher concentrations inhibit activity until almost complete inhibition is observed. Both the activation and the inhibition of cytochrome oxidase by thioridazine follow Michaelis-Menton kinetics with Vmax changing but Km remaining constant. The analysis of the 2 nm shift in the UV absorption spectrum of the thioridazine suggest that the binding of thioridazine to cytochrome oxidase involves multiple (535) binding sites on the enzyme with an average dissociation constant of 20 microM.     

Cytochrome oxidase induction after oxidative stress induced by adriamycin in liver of rats fed with dietary olive oil.

The influence of different kinds of dietary fat (8%) and of endogenous lipid peroxidation with regard to cytochrome c oxidase activity and cytochrome a + a3 concentrations in mitochondria from rat liver has been investigated. It was possible to confirm that the dietary fat induced higher phospholipid degradation in mitochondrial membranes; moreover an endogenous oxidative stress induced by adriamycin was able to increase the peroxidative effects. We have found that the peroxidative effects could sometimes induce an apparent enhancement of cytochrome oxidase activity due to a significant increase of cytochrome a + a3 content. This finding lets us suppose that both changes in the lipid environment and some peroxidation damage could occur in the membrane as a consequence of the fat assumed. Furthermore we should suggest that an induction of the synthesis of cytochrome a + a3 might be related to an enhanced production of peroxides at membrane level.     

Correlation of the kinetics of electron transfer activity of various eukaryotic cytochromes c with binding to mitochondrial cytochrome c oxidase.

1. A detailed study of cytochrome c oxidase activity with Keilin-Hartree particles and purified beef heart enzyme, at low ionic strength and low cytochrome c concentrations, showed biphasic kinetics with apparent Km1 = 5 x 10(-8) M, and apparent Km2 = 0.35 to 1.0 x 10(-6) M. Direct binding studies with purified oxidase, phospholipid-containing as well as phospholiptaining aid-depleted, demonstrated two sites of interaction of cytochrome c with the enzyme, with KD1 less than or equal to 10(-7) M, and KD2 = 10(-6) M. 2. The maximal velocities as low ionic strength increased with pH and were highest above ph 7.5. 3. The presence and properties of the low apparent Km phase of the kinetics were strongly dependent on the nature and concentration of the anions in the medium. The multivalent anions, phosphate, ADP, and ATP, greatly decreased the proportion of this phase and similarly decreased the amount of high affinity cytochrome c-cytochrome oxidase complex formed. The order of effectiveness was ATP greater than ADP greater than P1 and since phosphate binds to cytochrome c more strongly than the nucleotides, it is concluded that the inhibition resulted from anion interaction with the oxidase. 4mat low concentrations bakers' yeast iso-1, bakers' yeast iso-1, horse, and Euglena cytochromes c at high concentrations all attained the same maximal velocity. The different proportions of low apparent Km phase in the kinetic patterns of these cytochromes c correlated with the amounts of high affinity complex formed with purified cytochrome c oxidase. 5. The apparent Km for cytochrome c activity in the succinate-cytochrome c reductase system of Keilin-Hartree particles was identical with that obtained with the oxidase (5 x 10(-8) M), suggesting the same site serves both reactions. 6. It is concluded that the observed kinetics result from two catalytically active sites on the cytochrome c oxidase protein of different affinities for cytochrome c. The high affinity binding of cytochrome c to the mitochondrial membrane is provided by the oxidase and at this site cytochrome c can be reduced by cytochrome c1. Physiological concentrations of ATP decrease the affinity of this binding to the point that interaction of cytochrome c with numerous mitochondrial pholpholipid sites can competitively remove cytochrome c from the oxidase. It is suggested that this effect of ATP represents a possible mechanism for the control of electron flow to the oxidase.     

Fusion of phospholipid vesicles reconstituted with cytochromec oxidase and mitochondrial hydrophobic protein

Summary Reconstituted cytochrome oxidase liposomes were fused with liposomes reconstituted with mitochondrial hydrophobic protein, which acts as a membrane-bound uncoupler of cytochrome oxidase. Fusion was assayed by the loss of respiratory control of cytochrome oxidase as measured by the increased rate of ascorbate oxidation induced by hydrophobic protein when both proteins shared the same vesicles. Fusion was dependent on the presence of phosphatidylserine in the liposomes and Ca⁺⁺ in the aqueous medium. Phosphatidylcholine-phosphatidylserine liposomes required higher concentrations of phosphatidylserine and Ca⁺⁺ than did phosphatidylethanolamine-phosphatidylserine liposomes. Cytochrome oxidase vesicles containing high concentrations of phosphatidylserine showed little or no respiratory control, while those with lower concentrations showed high respiratory control; respiratory control could be induced by fusing cytochrome oxidase vesicles containing high phosphatidylserine with protein-free liposomes containing low phosphatidylserine concentration. If cytochrome oxidase vesicles and hydrophobic protein vesicles were prefused separately for 15 min, they lost the ability to fuse upon being subsequently mixed together. The reconstituted vesicles had diameters of about 200 Å; fusion yielded vesicles with diameters in excess of 1000 Å.     

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

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

Creatine kinase,energy-rich phosphates and energy metabolism in heart muscle of different vertebrates

Maximal activities of creatine kinase, pyruvate kinase and cytochrome oxidase and total concentrations of creatine and phosphorylated adenylates were measured in cardiac muscle of hagfish, eight teleost species, frog, turtle, pigeon and rat. The ratio of creatine kinase to cytochrome oxidase with cytochrome oxidase as a rough estimate of aerobic capacity and cellular “energy turnover”, was increased in myocardia of hagfish, turtle and crucian carp. These myocardia are likely to be frequently exposed to oxygen deficiency. In agreement with this, they possess a high relative glycolytic capacity as indicated by a high pyruvate kinase/cytochrome oxidase ratio. The creatine kinase/cytochrome oxidase ratio for the other myocardia varied within a factor of 2, except the value for cod myocardium which was below the others. Total creatine varied among species and was high in active species such as herring, pigeon and rat but also high in crucian carp. The variation in total concentration of phosphorylated adenylates was considerably less than the variation in total creatine. The high creatine kinase/ cytochrome oxidase ratio in myocardia likely to be challenged by hypoxia may represent an enhanced efficiency for both “spatial” and “temporal” buffering of phosphorylated adenylates to attenuate the impact of a depressed energy liberation. As to the differences in total creatine, this factor influences not only the cellular energy distribution but possibly also contractility via an effect on the free phosphate level.     

Responses of two protein-protein complexes to solvent stress: does water play a role at the interface?

We have analyzed the stability of the cytochrome c-cytochrome b5 and cytochrome c-cytochrome c oxidase complexes as a function of solvent stress. High concentrations of glycerol were used to displace the two equilibria. Glycerol promotes complex formation between cytochrome c and cytochrome b5 but inhibits that between cytochrome c and cytochrome c oxidase. The results with cytochrome b5 and cytochrome c were expected; the association of this complex is largely entropy driven. Our interpretation is that the cytochrome c-cytochrome b5 complex excludes water. The results with the cytochrome c oxidase and cytochrome c couple were not expected. We interpret them to mean that either glycerol is binding to the oxidase, thereby displacing the cytochrome c, or that water is required at this protein-protein interface. A requirement for substantial quantities of water at the interface of some protein complexes is logical but has been reported only once.     

Raman spectra of heme a, cytochrome oxidase-ligand complexes, and alkaline denatured oxidase.

We report 441.6 nm excitation resonance Raman spectra of oxidized and reduced monomeric heme a-imidazole, cytochrome oxidase-exogenous ligand complexes in various redox states, and alkaline denatured oxidase. These data show that, in reduced oxidase, the cytochrome a3 Raman spectrum has bands at 215, 364, 1230, and 1670 cm-1 not observed in the cytochrome a spectrum. The appearance of these bands in the reduced cytochrome a3 spectrum is due to interactions between the heme a of cytochrome a3 and its protein environment and not to intrinsic properties of heme a. These interactions are pH sensitive and strongly influence the vibrational spectra of both heme a groups. We assign the 1670-cm-1 band to the heme a formyl substituent and propose that the intensity of the 1670 cm-1 is high for reduced cytochrome a3 because the C==O lies in the porphyrin plane and is very weak for oxidized and reduced cytochrome a, oxidized cytochrome a3, and oxidized and reduced heme a-imidazole because the C==O lies out of the plane. We suggest that movement of the C==O in and out of the plane explains the ligand induced spectral shift in the optical absorption spectrum of reduced cytochrome a3. Finally, we confirm the observation of Adar & Yonetani (private communication) that, under laser illumination, resting oxidase is photoreactive.     

Biocytin hydrazide--a selective label for sialic acids, galactose, and other sugars in glycoconjugates using avidin-biotin technology

Biocytin hydrazide (BCHZ), a new, water-soluble, long-chained, biotin-containing hydrazide, was synthesized and used for the selective nonradioactive detection of glycoconjugates. Procedures were developed for labeling glycoconjugates on blots. The method involves either chemical (periodate-induced) or enzymatic (via galactose oxidase) oxidation of glycoconjugates, the resultant aldehyde groups are then labeled with biocytin hydrazide, followed by interaction with an avidin-based enzyme probe. Since the biotin-containing reagent is a relatively small, charged molecule, the primary labeling step may be carried out on intact cells and on membrane preparations as well as on blotted samples. On blots, the labeling pattern was similar for both periodate- and galactose oxidase-induced biotinylation procedures. In contrast, periodate-induced labeling of either erythrocyte membranes or cells (prior to blotting) produced an altered labeling pattern. Combined enzyme-induced biotinylation of membranes or cells resulted in a pattern similar to that observed for the direct staining of blots. Using galactose oxidase on human erythrocyte membranes, the procedure was sensitive enough to selectively label the Band 3 lactosaminoglycoprotein.     

Photosynthetic and respiratory electron transport in Cu2+-deficient Dunaliella

Growth inhibition of the green alga Dunalietla parva Lerche has been observed during cultivation in low Cu²⁺ media. A minimum endogenous Cu concentration for unrestricted growth of 100 to 200 nmol ml⁻¹ packed cell volume was estimated. At lower concentrations, Cu deficiency causes a decrease in photosynthesis and respiration. Assay of photosynthetic electron transport rates as well as the determination of several redox components showed that the target of Cu deprivation in the photosynthetic apparatus is the synthesis of Cu-containing plastocyanin. Consequently, inhibited formation of plastocyanin resulted in low activities of photosynthetic electron transport. A secondary, indirect effect of Cu deficiency is the reduction of thylakoid formation resulting in an additional decrease of photosynthesis compared to cultures with sufficient Cu²⁺.
The inhibitory influence of low Cu²⁺ on respiration was located at the site of cytochrome oxidase. In contrast to blue-green algae, a strong coordination of the biosynthesis of the cytochrome oxidase complex was evident. During restricted Cu²⁺ supply the formation of cytochiome aa₃ , another component besides Cu, was stalled. The resulting low activities of cytochrome oxidase are responsible for decreased respiratory electron transfer activity from NADPH to oxygen. At Cu²⁺ concentrations which exert only moderate effects on Dunalietla , the cytochrome oxidase reaction was more strongly affected than the photosystem I reaction.     

Ischemia, rather than reperfusion, inhibits respiration through cytochrome oxidase in the isolated, perfused rabbit heart: role of cardiolipin

Ischemia and reperfusion result in mitochondrial dysfunction, with decreases in oxidative capacity, loss of cytochrome c, and generation of reactive oxygen species. During ischemia of the isolated perfused rabbit heart, subsarcolemmal mitochondria, located beneath the plasma membrane, sustain a loss of the phospholipid cardiolipin, with decreases in oxidative metabolism through cytochrome oxidase and the loss of cytochrome c. We asked whether additional injury to the distal electron chain involving cardiolipin with loss of cytochrome c and cytochrome oxidase occurs during reperfusion. Reperfusion did not lead to additional damage in the distal electron transport chain. Oxidation through cytochrome oxidase and the content of cytochrome c did not further decrease during reperfusion. Thus injury to cardiolipin, cytochrome c, and cytochrome oxidase occurs during ischemia rather than during reperfusion. The ischemic injury leads to persistent defects in oxidative function during the early reperfusion period. The decrease in cardiolipin content accompanied by persistent decrements in the content of cytochrome c and oxidation through cytochrome oxidase is a potential mechanism of additional myocyte injury during reperfusion.     

COX23, a homologue of COX17, is required for cytochrome oxidase assembly

Deletion of reading frame YHR116W of the Saccharomyces cerevisiae nuclear genome elicits a respiratory deficiency. The encoded product, here named Cox23p, is shown to be required for the expression of cytochrome oxidase. Cox23p is homologous to Cox17p, a water-soluble copper protein previously implicated in the maturation of the Cu(A) center of cytochrome oxidase. The respiratory defect of a cox23 null mutant is rescued by high concentrations of copper in the medium but only when the mutant harbors COX17 on a high copy plasmid. Overexpression of Cox17p by itself is not a sufficient condition to rescue the mutant phenotype. Cox23p, like Cox17p, is detected in the intermembrane space of mitochondria and in the postmitochondrial supernatant fraction, the latter consisting predominantly of cytosolic proteins. Because Cox23p and Cox17p are not part of a complex, the requirement of both for cytochrome oxidase assembly suggests that they function in a common pathway with Cox17p acting downstream of Cox23p.     

The role of subunit III in bovine cytochrome c oxidase. Comparison between native, subunit III-depleted and Paracoccus denitrificans enzymes

In order to obtain information on the role of subunit III in the function and aggregation state of cytochrome c oxidase, the kinetics of ferrocytochrome c oxidation by the bovine cytochrome c oxidase depleted of its subunit III were studied and compared with those of the oxidase isolated from P. denitrificans which contains only two subunits. The aggregation state of both enzymes dispersed in dodecyl maltoside was also compared. The two-subunit oxidase from P. denitrificans gave linear Eadie-Hofstee plots and the enzyme resulted to be monomeric (Mr = 82 000) both, in gel filtration and sucrose gradient centrifugation studies. The bovine heart subunit III depleted enzyme, under conditions when the P. denitrificans cytochrome c oxidase was in the form of monomers, was found to be dimeric by sucrose gradient centrifugation analysis. At lower enzyme concentrations monomers were, however, detected by gel filtration. Depletion of subunit III was accompanied by the loss of small polypeptides (VIa, VIb and VIIa) and of almost all phospholipid (1-2 molecules were left per molecule of enzyme). The electron-transfer activity of the subunit III-depleted enzyme showed a monophasic Eadie-Hofstee plot, which upon addition of phospholipids became non-linear, similar to that of the control bovine cytochrome c oxidase. One of the roles of subunit III may be that of stabilising the dimers of cytochrome c oxidase. Lack of this subunit and loss of phospholipid is accompanied by a change in the kinetics of electron transfer, which might be the consequence of enzyme monomerisation.     

Measurement of free radical oxygen generation by cytochrome c reduction requirement for cytochrome c oxidase blockade

Data is presented showing that one commercial preparation of cytochrome c, used to trap and measure free radical superoxide anion, can be contaminated with cytochrome c oxidase activity. This activity can vary from lot to lot, can introduce variability into the measurement of superoxide anion and can result in falsely low estimations of free radical formation. This cytochrome c oxidase activity can be inhibited by low (0.2 mM) concentrations of KCN. Blockade of the cytochrome c oxidase activity allows reproducible measurement of superoxide anion formation at low levels by red cells.     

Palmitate decreases proton pumping of liver-type cytochrome c oxidase.

The H+/e- stoichiometry of reconstituted cytochrome c oxidase from bovine kidney, containing subunit VIaL (liver type), is 0.5 under standard conditions but 1.0 on addition of 1% cardiolipin to the lipid mixture (asolectin). Low concentrations of palmitate (half-maximal effect at 0.5 microm), but not laurate, myristate, stearate, oleate, 1-hexadecanol, palmitoyl glycerol and palmitoyl CoA, decreased the H+/e- ratio in the presence of cardiolipin from 1.0 to 0.5, accompanied by an increase of coupled, but not of uncoupled respiration of proteoliposomes. Cardiolipin and palmitate did not influence the H+/e- stoichiometry and respiration of reconstituted cytochrome c oxidase from bovine heart, containing subunit VIaH (heart-type). The H+/e- stoichiometry of the heart enzyme, however, is decreased from 1.0 to 0.5 by 5 mm intraliposomal ATP (instead of 5 mm ADP). It is assumed that palmitate binds to subunit VIaL. The partial uncoupling of proton pumping in cytochrome c oxidase is suggested to participate in mammalian thermogenesis.