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PubMed1982

Crystallization of mitochondrial cytochrome oxidase.

Cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was purified from beef heart mitochondria. By washing the oxidase with detergent on a hydrophobic interaction column, phospholipids were depleted to the level of 1 mol of cardiolipin per mol of heme a. Hydrophobic impurities and partially denatured oxidase were separated from the intact oxidase on an affinity column with cytochrome c as the specific ligand. The final preparation of the oxidase contained seven distinct polypeptides. The molecular weight of the oxidase was estimated to be 130,000 from its specific heme a and copper content and from the subunit composition. Crystals of the oxidase were obtained by slow removal of the detergent from the buffer in which the oxidase was dissolved. The needle-shaped crystals were 100 microns in average length and 5 microns in width, and they strongly polarized visible light. Electron diffraction patterns were obtained with an unstained glutaraldehyde-fixed single crystal by electron microscopy using 1,000-kV electrons. From electron micrographs and the diffraction patterns of the crystal, it was concluded that the crystal is monoclinic in the space group P21, with unit cell dimensions a = 92 A, b = 84 A, and c = 103 A, and alpha = beta = 90 degrees, gamma = 126 degrees.

Crystallography↗
PubMed1982

Kinetic characterization of the interaction between cytochrome oxidase and cytochrome c.

The mechanism of electron transfer catalyzed by cytochrome oxidase was investigated by monitoring the reaction of cytochrome oxidase with cytochrome c under carefully controlled anaerobic conditions. The kinetics of the reaction were examined by varying conditions of ionic strength, inhibitor binding, and oxidation-reduction potential. An analogue of cytochrome c in which the iron atom was replaced with cobalt was used to probe the effect of redox potential on the reaction. Under conditions of low ionic strength, there is very rapid oxidation of cytochrome c and reduction of oxidase which occurs at a rate of 3 X 10(7) M-1 s-1. The number of electrons transferred exhibit a hyperbolic dependence on the concentration of cytochrome c reaching a maximum of 2 electrons transferred at the highest concentration of reduced cytochrome c employed. The total number of electrons transferred was always observed to be distributed equally between cytochrome a and a second acceptor which appears to be the associated copper center; electron transfer to cytochrome a3 did not occur in the absence of oxygen. Substitution of cytochrome c by the cobalt analogue (which represents a decrease in oxidation-reduction potential of about 400 mV) yielded identical results indicating that the origin of the lack of reactivity of cytochrome a3 is of a kinetic nature. The effect of increasing the ionic strength on the reaction was 2-fold: a marked decrease in reaction rate and the appearance of biphasic kinetics with the amplitude of the very fast absorbance changes at 605 nm decreasing from 80% to 40% of the total anticipated from static absorbance measurements. Each of the two phases accounted for a maximum of 1 electron at the highest ionic strength employed. These results are simulated in terms of a sample kinetic reaction scheme involving a two-step electron transfer at one binding site.

Anaerobiosis↗
PubMed1993

Protons, pumps, and potentials: control of cytochrome oxidase.

Cytochrome c oxidase oxidizes cytochrome c and reduces molecular oxygen to water. When the enzyme is embedded across a membrane, this process generates electrical and pH gradients, and these gradients inhibit enzyme turnover. This respiratory control process is seen both in intact mitochondria and in reconstituted proteoliposomes. Generation of pH gradients and their role in respiratory control are described. Both electron and proton movement seem to be implicated. A topochemical arrangement of redox centers, like that in the photosynthetic reaction center and the cytochrome bc1 complex, ensures charge separation as a result of electron movement. Proton translocation does not require such a topology, although it does require alternating access to the two sides of the membrane by proton-donating and accepting groups. The sites of respiratory control within the enzyme are discussed and a model presented for electron transfer and proton pumping by the oxidase in the light of current knowledge of the transmembranous location of the redox centers involved.

Animals↗
PubMed1991

Intrinsic tryptophan phosphorescence as a marker of conformation and oxygen diffusion in purified cytochrome oxidase.

Cytochrome oxidase exhibits phosphorescence from tryptophan in aqueous solution in the absence of oxygen. The lifetime for the resting reduced enzyme suspended in Tween-20 is around 30 ms at pH 8. The lifetime is longest between pH 7 and 8 and decreases with lowering of pH. Oxygen quenches the phosphorescence with a Stern-Volmer quenching constant of approximately 5 x 10(7) M-1.s-1 at 5 degrees C whereas cytochrome c has no effect. We interpret these results to indicate that room temperature tryptophan phosphorescence arises from tryptophan(s) in structured region(s) remote from the hemes and that the protein does not impose a significant barrier for the diffusion of oxygen.

Animals↗
PubMed1986

Effect of saturated phosphatidylcholines on the functional properties of reconstituted cytochrome oxidase.

Cytochrome oxidase was incorporated into liposomes, at various protein/lipid ratios, composed of either a phosphatidylcholine of varying chain length and symmetry or asolectin. Catalytic activity and respiratory control were assayed at two temperatures. All preparations showed higher activity at low protein/lipid ratios, but only asolectin showed respiratory control. A spectroscopic determination of the vectorial orientation of oxidase molecules showed that, for proteoliposomes with saturated lipids, 100% of oxidase molecules could be reduced by external substrate as compared with 75% for asolectin proteoliposomes. Freeze-fracture electron microscopy confirmed that oxidase was incorporated into these proteoliposomes and differential scanning calorimetry indicated that the protein induces significant disruption in the long range packing of the saturated phospholipids. We propose that the oxidase molecules in proteoliposomes formed from saturated phosphatidylcholines do not display respiratory control because they are unable to assume the transmembrane orientation necessary for full vectorial activity.

Animals↗
PubMed1979

Ultrastructural demonstration of cytochrome oxidase via cytochrome C in cerebral cortex.

A procedure for the ultrastructural cytochemical localization of cytochrome oxidase via cytochrome c in the cerebral cortex is described. Vascular perfusion fixation by formaldehyde and glutaraldehyde of different concentrations and mixtures of the two gave varying results. A mixture of 4% formaldehyde and 0.5% glutaraldehyde gave the best combination of ultrastructural preservation and retention of enzyme activity. Histochemical methods were examined for optimum incubation conditions, based on the oxidative polymerization of 3,3'-diaminobenzidine (DAB) to an osmiophilic product. The reaction product was discretely localized within intercristate and the intermembrane space of mitochondria. The staining pattern was the same in nerve cells and in neuroglia and their processed. The DAB reaction product was also found in mitochondria of the endothelial cells.

3,3'-Diaminobenzidine↗
PubMed1975

Studies on cytochrome oxidase. Interactions of the cytochrome oxidase protein with phospholipids and cytochrome c.

1. By the application of the principle of the sequential fragmentation of the respiratory chain, a simple-method has been developed for the isolation of phospholipid-depleted and phospholipid-rich cytochrome oxidase preparations. 2. The phospholip-rich oxidase contains about 20% lipid, including mainly phosphatidylethanolamine, phosphatidylcholine, and cardiolipin. Its enzymic activity is not stimulated by an external lipid such as asolectin. 3. The phospholipid-depleted oxidase contains less than 0.1% lipid. It is enzymically inactive in catalyzing the oxidation of reduced cytochrome c by molecular oxygen. This activity can be fully restored by asolectin; and partially restored (approximately 75%) by purified phospholipids individually or in combination. The activity can be partially restored also by phospholipid mixtures isolated from mitochondria, from the oxidase itself, and from related preparations. Among the detergents tested only Emasol-1130 and Tween 80 show some stimulatory activity. 4. The phospholipid-depleted oxidase binds with cytochrome c evidently by "protein-protein" interactions as does the phospholipid-rich or the phospholipid-replenished oxidase to form a complex with the ratio of cytochrome c to heme a of unity. The complex prepared from phospholipid-depleted cytochrome oxidase exhibits a characteristic Soret absorption maximum at 415 nm in the difference spectrum of the carbon monoxide-reacted reduced form minus the reduced form. This 415-nm maximum is abolished by the replenishment of the complex with a phospholipid or by the dissociation of the complex in cholate or in a medium of high ionic strength. When ascorbate is used as an electron donor, the complex prepared from phospholipid-depleted cytochrome oxidase does not cause the reduction of cytochrome a3 which is in dramatic contrast to the complex from the phospholipid-rich or the phospholipid-replenished oxidase. However, dithionite reduces cytochrome a3 in all of the preparations of the cytochrome c-cytochrome oxidase complex. These facts suggest that the action of phospholipid on the electron transfer in cytochrome oxidase may be at the step between cytochromes a and a3. This conclusion is substantiated by preliminary kinetic results that the electron transfer from cytochrome a to a3 is much slower in the phospholipid-depleted than in phospholipid-rich or phospholipid-replenished oxidase. On the basis of the cytochrome c content, the enzymic activity has been found to be about 10 times higher in the system with the complex (in the presence of the replenishedhe external medium unless energy is provided, and that

Animals↗
PubMed1994

Circular dichroism studies of the binding of mammalian and non-mammalian cytochromes c to cytochrome c oxidase, cytochrome c peroxidase, and polyanions.

The effects of binding of Candida krusei, Drosophila melanogaster, horse, human, and rat cytochromes c to beef cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, EC 1.9.3.1) and yeast cytochrome c peroxidase (ferricytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) on their circular dichroism spectra were determined. The binding to cytochrome oxidase results in a positive increase in the ellipticities of the positive and negative Cotton effects at 404 nm and 417 nm of cytochrome c. The horse, human, and rat cytochromes c display less of an increase in the ellipticity of the positive Cotton effect at 404 nm, but more of a positive change in the negative Cotton effect at 417 nm than the C. krusei or D. melanogaster proteins. Interaction with yeast cytochrome c peroxidase elicits only a positive change in the ellipticity of the positive Cotton effect at 404 nm. No significant change is observed in the negative Cotton effect at 417 nm. Rat cytochrome c variants with a phenylalanine in place of tyrosine-67 and/or an alanine in place of proline-30 all display circular dichroism spectral changes upon binding to cytochrome c oxidase or cytochrome c peroxidase identical to those of the unaltered protein. The increase in ellipticity at 404 nm upon binding occurs even though replacement of tyrosine-67 results in the loss of the positive Cotton effect at this position. Polyglutamate and phosvitin complexes of cytochrome c show changes in the circular dichroism spectrum similar to those observed with cytochrome c peroxidase. However, the magnitudes of the spectral changes were considerably less. A model is proposed in which the main cause of the circular dichroism spectral changes observed upon complexation arise from the exclusion of solvent from the exposed front heme edge. According to this model, the exclusion of solvent changes the relative asymmetry of the environment of the electronic transitions of the heme prosthetic group of cytochrome c, resulting in observed circular dichroic effects.

Animals↗
PubMed2002

Influence of structure, pH and membrane potential on proton movement in cytochrome oxidase.

Cytochrome c oxidase (CcO) reconstituted into phospholipid vesicles and subject to a membrane potential, exhibits different characteristics than the free enzyme, with respect to effects of mutations, pH, inhibitors, and native structural differences between CcO from different species. The results indicate that the membrane potential influences the conformation of CcO and the direction of proton movement in the exit path. The importance of the protein structure above the hemes in proton exit, back leak and respiratory control is discussed.

Biological Transport, Active↗
PubMed1980

Crystallization of part of the mitochondrial electron transfer chain: cytochrome c oxidase--cytochrome c complex.

Cytochrome c oxidase (ferrocytochrome c oxygen oxidoreductase, EC 1.9.3.1) was purified from beef heart mitochondria by affinity chromatography. Phospholipids were removed by washing the oxidase with detergent on the affinity column; 1 mole of cardiolipin remained per mole of heme a. The oxidase was mixed with excess cytochrome c in 1.5% (wt/vol) cholate to form a complex. Slow removal of the detergent from the mixture by dialysis resulted in crystallization of cytochrome oxidase in the form of a 1:1 complex with cytochrome c. The chemical composition and spectrophotometric properties of the crystal are described. Increasing the solubility of a hydrophobic membrane protein by combination with hydropholic ligand is demonstrated as a maneuver for crystallizing the membrane protein.

Animals↗
PubMedDate not supplied

[Activity and stability of NAD.H-oxidase, succinate oxidase and cytochrome oxidase of rat liver and pancreatic mitochondria in chronic allergic colitis].

A comparative study of thermostability of NADH-, succinate- and cytochrome oxidases was carried out in liver mitochondria and pancreas of rats in normal state and in chronic allergic colitis. In the pathology degradative alterations occurred in mitochondria, which were manifested as a decrease in the stability of mitochondrial mutinzyme systems of tissues studies. The results suggested occurrence of latent impairments in structure of mitochondria.

Animals↗
PubMed1980

A two-subunit cytochrome c oxidase (cytochrome aa3) from Paracoccus dentrificans.

Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, EC 1.9.3.1) was purified from the cytoplasmic membrane of the bacterium Paracoccus denitrificans. The enzyme contains two heme groups (a and a3) and two copper atoms per minimal unit, oxidizes mammalian cytochrome c at a high rate, and, when incorporated into liposomes, generates an electrochemical proton gradient during cytochrome c oxidation. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis reveals only two subunits of apparent molecular weights 45,000 and 28,000; they appear to correspond to the two largest mitochondrially made subunits of the seven-subunit cytochrome c oxidase isolated from yeast mitochondria. Because of its structural simplicity. Paracoccus cytochrome c oxidase offers new possibilities for exploring the mechanism of cytochrome c oxidase function.

Amino Acids↗
PubMed1993

Identification and characterization of the ctaC (coxB) gene as part of an operon encoding subunits I, II, and III of the cytochrome c oxidase (cytochrome aa3) in the cyanobacterium Synechocystis PCC 6803.

The gene (coxII = coxB = ctaC) encoding subunit II of Synechocystis PCC 6803 cytochrome c oxidase has been isolated by screening a genomic DNA library in pUC18 with a 17-bp oligonucleotide probe (probe C) derived from coxI of Paracoccus denitrificans after Southern blots with a 19-kb oligonucleotide (probe A) derived from coxII of P. denitrificans had given equivocal results. A 2.2 kb PstI-KpnI restriction fragment was subcloned into pUC 18 and the resulting plasmid pDAUV26, which contained the probe C-binding site near the downstream end was found also to contain the whole coxII gene upstream of this site. The novel plasmid pDAUV 26 was used to transform competent E. coli cells, propagated therein, and the sequence determined. The 2.2 kb insert contained the entire coding region for the coxII gene together with a GAG start codon, a TAA stop codon, and a putative Shine-Dalgarno sequence. The deduced COII polypeptide is composed of 319 aa (calculated molecular mass of 32,800) plus a N-terminal leader sequence of 20 aa. The hydropathy plot suggests two lipophilic transmembrane domains near the N-terminus connected with an extremely hydrophilic aa stretch on the cytosolic side, while an unusually long (> 50 aa) aa stretch on the periplasmic (= intrathylakoidal) side leads to a typical cyanobacterial threonine in place of the first conserved glutamate of the cytochrome c-binding region in all other COII proteins. Together with a considerably shortened and interrupted aromatic aa stretch in this region, these differences are discussed in terms of the peculiar affinity of cyanobacterial cytochrome oxidases for acidic c-type cytochromes. Other invariant features such as the strictly conserved CuA-binding aa, however, are found in correct positions.

Amino Acid Sequence↗