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PubMed1979

A temperature-induced absorption band centered in the region of 666 nm related to the configuration of the active site in frozen cytochrome oxidase.

The existence of a temperature-induced absorption band centred in the region of 666 nm is demonstrated for both membrane-bound and soluble cytochrome oxidase in the frozen state. The 666 nm band is generated solely by an increase in temperature of both fully reduced and mixed valence state cytochrome oxidase in the presence of CO or O2 within the 'pocket' containing the active site; it is not formed in the absence of both CO and O2 from the sample. The formation of the 666 nm band is entirely reversible when the temperature is decreased again and its formation is not dependent on the presence of liganded CO at the sixth coordination site of haem a3 in the low temperature range (below --120 degrees C) prior to photolysis. The shape and intensity of the 666 nm band are not affected by the extent of CO recombination following flash and photolysis and temperature increase and are not affected by changes in the valence states of the four metal centres when the O2 reaction is in progress.

Animals↗

PubMed1991

Endogenous cytochrome c and cytochrome oxidase in rat and mouse kidney: light microscopic histochemical study.

Activity of cytochrome c oxidase and the level of endogenous cytochrome c were investigated light microscopically in adult rat and mouse kidney by incubating unfixed frozen sections with diaminobenzidine (DAB) in the absence or presence of exogenous cytochrome c. The results suggest that DAB staining intensity mainly reflects the local density of mitochondria and only occasionally visualizes the differences in cytochrome oxidase activity and/or endogenous cytochrome c content. Most intense reaction was observed in proximal and distal tubules both in rat and mouse. Finer differentiation of reactivity in particular nephron segments and interspecies differences between rat and mouse kidney are also described.

3,3'-Diaminobenzidine↗

PubMed1980

Cytochrome oxidase rotates in the inner membrane of intact mitochondria and submitochondrial particles.

A transient dichroism is detected after photolysis by a linearly polarized laser flash of the cytochrome oxidaseCO complex in bovine heart mitochondria, rat heart mitochondria, and bovine heart submitochondrial particles. A decay in the absorption anisotropy is characterized by a time constant of about 300 to 400 mus in both mitochondria and submitochondrial particles. Since vesicle tumbling in the time range less than 5 ms can be excluded in these experiments, we conclude that cytochrome oxidase rotates in the mitochondrial membrane with a relaxation time of several hundred microseconds. However, it is likely that only about one-half of cytochrome oxidase contributes to the observed decay, the remainder being relatively immobile.

Animals↗

PubMed2003

Water-gated mechanism of proton translocation by cytochrome c oxidase.

Cytochrome c oxidase is essential for aerobic life as a membrane-bound energy transducer. O(2) reduction at the haem a(3)-Cu(B) centre consumes electrons transferred via haem a from cytochrome c outside the membrane. Protons are taken up from the inside, both to form water and to be pumped across the membrane (M.K.F. Wikström, Nature 266 (1977) 271; M. Wikström, K. Krab, M. Saraste, Cytochrome Oxidase, A Synthesis, Academic Press, London, 1981 ). The resulting electrochemical proton gradient drives ATP synthesis (P. Mitchell, Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin, UK, 1966 ). Here we present a molecular mechanism for proton pumping coupled to oxygen reduction that is based on the unique properties of water in hydrophobic cavities. An array of water molecules conducts protons from a conserved glutamic acid, either to the Delta-propionate of haem a(3) (pumping), or to haem a(3)-Cu(B) (water formation). Switching between these pathways is controlled by the redox-state-dependent electric field between haem a and haem a(3)-Cu(B), which determines the water-dipole orientation, and therefore the proton transfer direction. Proton transfer via the propionate provides a gate to O(2) reduction. This pumping mechanism explains the unique arrangement of the metal cofactors in the structure. It is consistent with the large body of biochemical data, and is shown to be plausible by molecular dynamics simulations.

Electron Transport↗

PubMed1996

Color, orientation and cytochrome oxidase reactivity in areas V1, V2 and V4 of macaque monkey visual cortex.

Color and orientation processing in the macaque monkey first segregates into cytochrome oxidase (CO)-rich blobs and -poor interblobs of area V1, from where the two streams flow through areas V2 and V4. This parallel representation is believed to enhance processing speed by compartmentalizing tasks of similar kinds, though our knowledge of the mechanisms is still elementary. We have examined the interaction and separation of color and orientation processing in neurons (n = 569) of the macaque visual cortex (V1, V2, V4) on the basis of microelectrode recordings. In all three areas, neurons selective for midspectral (MS) colors (e.g., yellow, green) were also found to be more orientation selective than those preferring endspectral (ES) colors (e.g., blue, red). The majority of achromatic (AC) cells responsive to bright stimuli were also orientation selective. When locations of cells and penetration columns were correlated with cytochrome oxidase (CO) landmarks in V1 and V2, V1 interblob and V2 interstripe cells were found to be predominantly midspectral and oriented, while V1 blob and V2 thin stripe cells were found to be predominantly endspectral and non-oriented. Cells preferring dark colors were found to cluster in thick stripes in V2, and in columns in V4. Separate clustering of midspectral (MS) and endspectral (ES) systems in V4 was also noted. With the results shown in a companion paper (Behav. Brain Res., 76 (1996) 51-70), the present data indicate that the visual system appears to optimize color and spatial acuity by separating chromatic information into non-oriented endspectral and oriented midspectral components.

Action Potentials↗

PubMed1981

Reaction of Mixed Valence State Cytochrome Oxidase with Oxygen in Plant Mitochondria: A STUDY BY LOW TEMPERATURE FLASH PHOTOLYSIS AND RAPID WAVELENGTH SCANNING OPTICAL SPECTROMETRY.

The reaction of mixed valence state cytochrome oxidase (Cu(A) (2+)a(3+) . Cu(B) (+)a(3) (2+)) with O(2) at 173 K has been investigated in purified potato mitochondria by low temperature flash photolysis and rad wavelength scanning optical spectrometry in the visible region. The kinetics of the reaction have been analyzed simultaneously at six wavelength pairs (586-630, 590-630, 594-630, 604-630, 607-630, and 610-630 nanometers) by nonlinear optimization techniques, and found to proceed by a two-species sequential mechanism. The "pure" difference spectra of the two species, I(M) and II(M), relative to unliganded mixed valence state cytochrome oxidase have been obtained. The difference spectrum of species I(M) is characterized by a peak at 591 nanometers, with a shoulder at 584 nanometers and a trough at 602 nanometers, and that of species II(M) by an alpha band split into a prominent peak at 607 nanometers and a small side peak at 594 nanometers. Evidence is presented to suggest that these two bands arise from O(2) (-) --> Cu(B) (2+) and O(2) (-) --> a(3) (2+) charge transfer transitions which would imply that O(2) (-) forms a bridging ligand between Cu(B) and the iron atom of cytochrome a(3) in species II(M). The kinetics of the reaction and the spectral characteristics of species I(M) and II(M) obtained with the potato mitochondrial system are compared and contrasted with data in the literature on the beef heart mitochondrial system.

Journal Article↗

PubMed1989

Increased cytochrome oxidase activity in the diabetic rat retinal pigment epithelium.

We have investigated the effects of diabetes on retinal oxidative metabolism. Since activity of the mitochondrial enzyme, cytochrome oxidase, has been demonstrated to be a reliable indicator of oxidative metabolism and physiological activity, we used cytochemical techniques to study the activity of this enzyme in spontaneously diabetic, streptozotocin-diabetic, and control rat retinas. Light microscope results showed an increase in staining for cytochrome oxidase activity in the diabetic RPE cell layer as compared with the control. Quantitative electron microscope analysis showed a significant increase in RPE cells with highly reactive mitochondria as compared with the controls. Mitochondrial staining within the diabetic photoreceptor and retinal vascular endothelial cells was normal. RPE cell volume and surface area, as well as number and volume of mitochondria, were unchanged. This increase in oxidative enzyme activity is further evidence of RPE cell alteration in diabetes.

Animals↗

PubMed1987

The reaction of cytochrome oxidase with cyanide. Preparation of the rapidly reacting form and its conversion to the slowly reacting form.

The magnitude of the slow phase of reaction of cytochrome oxidase with cyanide has been correlated with the size of the epr signal at g' = 12. This epr signal was not found in submitochondrial particles, and significant g' = 12 epr was only observed late in the purification of solubilized enzyme. The Hartzell-Beinert procedure for the purification of cytochrome oxidase (Hartzell, C.R., and Beinert, H. (1974) Biochim. Biophys. Acta 368, 318-338) has been modified so that the purified enzyme reacts in a single rapid phase with potassium cyanide and lacks the g' = 12 epr signal. This enzyme could be converted to the slowly reacting form upon incubation at low pH and/or low enzyme concentration. No procedure for the stable reversal of the process could be found. Some physical and chemical properties of the two forms of the enzyme are compared.

Cyanides↗

PubMed1980

The subunit composition of mammalian cytochrome c oxidase.

Cytochrome c oxidase from rat liver mitochondria was separated into 12 different protein subunits by application of a highly resolving sodium dodecylsulfate/gel electrophoretic system of different compositions. The 12 protein subunits are shown to represent integral components of mammalian type cytochrome c oxidase for the following reasons. 1. All 12 subunits copurify through various purification procedures. 2. The subunit composition of the isolated enzyme is identical to that of the immunoprecipitated one. 3. All 12 subunits are present in the complex at one to one stoichiometric amounts. 4. A similar composition of 12 subunits was also found for cytochrome c oxidase from rat kidney, pig heart, rabbit liver and stone-marten liver. The difference between our results and all other published data on the subunit composition of mammalian-type cytochrome c oxidase, based on gel electrophoretic analysis, is due to the insufficient resolving power of previously used gel systems and the very similar molecular weight of subunits VIa, b, c, and VIIa, b, c.

Animals↗

PubMed2001

Cytochrome oxidase activity in splanchnic organs of portal hypertensive rats.

OBJECTIVE: Portal hypertension is characterized by hyperdynamic splanchnic circulation associated with the development of portosystemic portal collateral circulation. Since blood flow regulation mechanisms in the splanchnic organs can be metabolic, its metabolic capacity has been studied using the mitochondrial enzyme cytochrome C oxidase as histochemical marker. METHOD: Cytochrome oxidase was quantified with a histochemical technique in the liver, pancreas and small bowel of Wistar rats in the control group (n = 8) and in rats with portal hypertension by triple stenosing ligation of the portal vein (n = 9) at 28 days of evolution. RESULTS: All rats with portal hypertension develop portosystemic collateral circulation. In these animals, cytochrome oxidase activity increases (p < 0.01) in the liver (left lateral lobe, periportal zone: 91.81 +/- 5.18 vs. 86.03 +/- 2.82) exocrine pancreas (125.6 +/- 7.25 vs 117.57 +/- 6.43; p < 0.05) as well as in the mucosa (crypts) and duodenum serosa, jejunum and ileum while it decreases in the pericentral zone of the hepatic acinus and intestinal villi. CONCLUSION: Cytochrome oxidase is considered an endogenous marker of local tissular metabolic capacity, so that its increased activity in the small bowel mucosa, crypts, exocrine pancreas and visceral peritoneum may be a metabolic factor that induces splanchnic hyperdynamic circulation in short-term portal hypertensive rats.

Animals↗

PubMed1993

Morphometry of rat olfactory bulbs stained for cytochrome oxidase reveals that the entire population of glomeruli forms early in the neonatal period.

Cytochrome oxidase staining selectively highlights the synapse-rich neuropil of olfactory bulb glomeruli. Detailed morphometric enumeration of glomeruli in cytochrome oxidase-stained sections from postnatally developing rats reveals that the entire adult population of glomeruli (2400/bulb) forms early in life, the process being complete by days 3-5 postnatal. Newborn's glomeruli range in diameter from a mean of about 50 microns to a maximum of about 70 microns and undergo a 3-fold increase in diameter and a 20-fold increase in volume during days 1-50 postnatal. The presence of the entire adult's glomeruli in the neonate calls for a serious revision of some current views on glomerular development and, given the critical roles of glomeruli as modules, the findings bear important implications for the organization of olfactory system and early development of olfactory functions.

Animals↗

PubMed1992

The dioxygen cycle. Spectral, kinetic, and thermodynamic characteristics of ferryl and peroxy intermediates observed by reversal of the cytochrome oxidase reaction.

The catalytic mechanism of O2 reduction by cytochrome oxidase was studied in isolated mitochondria and mitoplasts by partial reversal of the reaction. At a high redox potential (Eh) of cytochrome c, high pH, and a high electrochemical proton gradient (delta mu H+) across the inner mitochondrial membrane, the initial ferriccupric state (O) of the oxidized enzyme's bimetallic oxygen reaction center is converted to ferryl (F) and peroxy (P) intermediates, the optical spectroscopic properties of which are reported in detail. This is associated with reversed electron transfer from the bimetallic center to ferricytochrome c. The kinetics of reduction of ferricytochrome c by the reversed electron transfer process are compared with the kinetics of formation of F and P. The results are consistent with transfer of one electron from the ferric-cupric bimetallic center (O) to cytochrome c, yielding the F intermediate, followed by transfer of one electron from the latter to cytochrome c, yielding the P state. In the absence of an effective redox buffer, poising cytochrome c highly oxidized, these primary events are immediately followed by reoxidation of cytochrome c, which is ascribed to forward electron transfer to enzyme molecules still in the O state. This forward reaction also results in accumulation of the P intermediate. Kinetic stimulations of the data predict equilibrium constants for the reversed electron transfer steps, and Em,7 values of approximately 1.1 and 1.2 V may be calculated for the F/O and P/F redox couples, respectively, at delta mu H+ and delta psi equal to zero. Taken together with previously measured Em,7 values, these data indicate that it is the two-electron reduction of bound dioxygen to bound peroxide that is responsible for the irreversibility of the catalytic dioxygen cycle of cell respiration.

Animals↗

PubMed2002

Comparison between the nitric oxide reductase family and its aerobic relatives, the cytochrome oxidases.

The denitrification pathway has been studied in the hyperthermophilic archaeon Pyrobaculum aerophilum. In contrast with Gram-negative bacteria, all four denitrification enzymes are membrane-bound. P. aerophilum is also the only denitrifyer identified so far in which menaquinol is the electron donor to all four denitrification reductases. The NO reductase (NOR) of P. aerophilum belongs to the superfamily of haem-copper oxidases and is of the qNOR (quinol-dependent) type. Three types of NOR have been purified so far: cNOR (cytochrome c/pseudoazurin-dependent), qNOR and qCu(A)NOR (qNOR that contains Cu(A) at the electron entry site). It is proposed that the NORs and the various cytochrome oxidases have evolved by modular evolution, in view of the structure of their electron donor sites. qNOR is further proposed to be the ancestor of all NORs and cytochrome oxidases belonging to the superfamily of haem-copper oxidases.

Aerobiosis↗

PubMed1992

Isolation and partial purification of cytochrome oxidases from Bacillus thuringiensis subsp. israelensis HD-567.

Analyses of the cell membrane fractions by spectral absorbance revealed the presence of cytochrome c and three cytochrome oxidases in Bacillus thuringiensis subsp. israelensis HD-567-cytochromes a+a3, d, and o. A modified procedure was used to purify the cytochrome c:o complex from this organism. The oxidase complex was first solubilized from a sonic-disrupted cell membrane fraction (R3 fraction) using deoxycholate and KCl. The resulting soluble fraction was further purified by Sephadex G-50 gel filtration and DEAE ionexchange chromatography. TMPD oxidase specific activity and cytochrome concentration were assayed to monitor the purification procedures. The F7 fraction (obtained after G-50 chromatography) contained cytochrome c (0.44 nmole/mg protein), cytochrome a+a3 (0.26 nmole/mg protein), and cytochrome o (0.38 nmole/mg protein), which had 6-fold, 3.4-fold and 18.9-fold increase, respectively, by comparison with the original R3 fraction. The TMPD oxidase specific activity of the F7 fraction also increased 4.8 fold. The F8 fraction (obtained after the final DEAE chromatography) contained a cytochrome c:o complex only (cytochrome c, 0.46 nmole/mg protein; cytochrome o, 0.16 nmole/mg protein), but no cytochrome a+a3 was found. Both the TMPD oxidase specific activity and the cytochrome o were attenuated greatly in comparison with the F7 fraction. SDS-PAGE analysis revealed that the F7 fraction contained numerous protein components, while the F8 fraction contained only a prominent major protein of 113.5 kD thought to be the cytochrome c:o complex, and a minor polypeptide (MW = 65.8 kD). Although the final DEAE procedure removed many undesired polypeptides, TMPD oxidase activity and cytochrome o component were also lost greatly. Kinetic studies of this cytochrome c:o complex is in progress in our laboratory.

Bacillus thuringiensis↗

PubMed1980

Isolation, subunit composition, and site of synthesis of human cytochrome c oxidase.

Cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1), the terminal oxidase of the respiratory chain in eucaryotic cells, has been purified from human placenta mitochondria. Seven polypeptides have been identified reproducibly by high-resolution electrophoresis of the enzyme complex through sodium dodecyl sulfate (Na-DodSO4)--urea polyacrylamide gels; these correspond closely in size to the subunits of beef heart cytochrome c oxidase. When HeLa cells, grown in suspension culture, were pulse-labeled with [35S]methionine in the presence of cycloheximide to inhibit cytoplasmic protein synthesis and chased with an excess of unlabeled methionine in the absence of the drug, the mitochondrially synthesized polypeptides were resolved into at least 17 components by NaDodSO4--urea polyacrylamide gel electrophoresis. After labeled HeLa mitochondria were mixed with human placenta mitochondria and the cytochrome c oxidase was isolated, three of the labeled components were found to copurify with the three largest subunits of the complex. We conclude that human cytochrome c oxidase contains seven subunits, the three largest of which are synthesized on mitochondrial ribosomes, while the other four are synthesized in the cytoplasm.

Electron Transport Complex IV↗

PubMed1998

Oxidation and reduction of cytochrome oxidase in the neonatal brain observed by in vivo near-infrared spectroscopy.

Near-infrared spectroscopy was used to determine the relationship between the redox state of mitochondrial cytochrome oxidase CuA and haemoglobin oxygenation in the isoflurane-anaesthetized neonatal pig brain. Adding 7% CO2 to the inspired gases increased the total haemoglobin concentration by 8 microM and oxidized CuA by 0.2 microM. Decreasing the inspired oxygen fraction to zero for 90 s dropped the oxyhaemoglobin concentration by 27 microM and reduced CuA by 1.8 microM. However, no change in the CuA redox state was observed until oxyhaemoglobin had decreased by more than 10 microM. The response of the CuA redox state to these stimuli was very similar following 80% replacement of the haemoglobin by a perfluorocarbon blood substitute; this demonstrates that the results in the normal haematocrit were not a spectral artefact due to the high haemoglobin/cytochrome oxidase ratio. We conclude that the large reductions in the CuA redox state during anoxia are caused by a decrease in the rate of oxygen delivery to the cytochrome oxidase oxygen binding site; the small oxidations, however, are likely to reflect the effects of metabolic changes on the redox state of CuA, rather than increases in the rate of oxygen delivery.

Animals↗

PubMed1988

Chemical modification of the CuA site affects the proton pumping activity of cytochrome c oxidase.

Cytochrome c oxidase in which the CuA site has been perturbed by extensive modification of the enzyme with the thiol reagent p-(hydroxymercuri)benzoate has been reconstituted into phospholipid vesicles. The reconstituted vesicles lack respiratory control, and the orientation of the enzyme in the vesicles is similar to that of the native cytochrome c oxidase. In the proton translocation assay, the vesicles containing the modified enzyme behave as if they are unusually permeable to protons. When the modified and native proteins were coreconstituted, a substantial portion of the latter became uncoupled as revealed by low respiratory control and low overall proton pumping activity. These results suggest that the modified enzyme catalyzes a passive transport of protons across the membrane. When milder conditions were used for the chemical modification, a majority of the thiols reacted while the CuA site remained largely intact. Reconstitution of such a partially modified cytochrome c oxidase produced vesicles with respiratory control and proton translocating activity close to those of reconstituted native enzyme. It thus appears that the appearance of a proton leak is related to the perturbation of the CuA site. These observations suggest that the structure of CuA may be related to the role of this site in the proton pumping machinery of cytochrome c oxidase.

Animals↗

PubMed1975

The ultrastructural cytochemistry of lactic dehydrogenase, succinic dehydrogenase, dihydro-nicotinamide adenine dinucleotide diaphorase and cytochrome oxidase activities in hair cell mitochondria of the guinea pig cochlea.

The use of cinnamyl nitroblue tetrazolium chloride (DS-NBT) in dehydrogenase experiments (lactic dehydrogenase, succinic dehydrogenase, nicotinamide adenine dinucleotide diaphorase) and 3,3'-diaminobenzidine tetrahydrochloride (DAB) in cytochrome oxidase experiments indicated that mitochondrial oxidoreduction reactions from nicotinamide adenine dinucleotide to cytochrome oxidase are located on the inner mitochondrial membrane in the outer compartment and the intracristate spaces. These reactions behave according to the chemiosmotic hypothesis. The cochlear hair cell mitochondria are cytochemically indistinguishable from free liver mitochondria. The heterogeneous mitochondrial staining pattern is related to the osmolarity of the incubation media, solubility of the enzymes and pH of the medium, but not to the fixation method.

Animals↗