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PubMed2001

On the role of the K-proton transfer pathway in cytochrome c oxidase.

Cytochrome c oxidase is a membrane-bound enzyme that catalyzes the four-electron reduction of oxygen to water. This highly exergonic reaction drives proton pumping across the membrane. One of the key questions associated with the function of cytochrome c oxidase is how the transfer of electrons and protons is coupled and how proton transfer is controlled by the enzyme. In this study we focus on the function of one of the proton transfer pathways of the R. sphaeroides enzyme, the so-called K-proton transfer pathway (containing a highly conserved Lys(I-362) residue), leading from the protein surface to the catalytic site. We have investigated the kinetics of the reaction of the reduced enzyme with oxygen in mutants of the enzyme in which a residue [Ser(I-299)] near the entry point of the pathway was modified with the use of site-directed mutagenesis. The results show that during the initial steps of oxygen reduction, electron transfer to the catalytic site (to form the "peroxy" state, P(r)) requires charge compensation through the proton pathway, but no proton uptake from the bulk solution. The charge compensation is proposed to involve a movement of the K(I-362) side chain toward the binuclear center. Thus, in contrast to what has been assumed previously, the results indicate that the K-pathway is used during oxygen reduction and that K(I-362) is charged at pH approximately 7.5. The movement of the Lys is proposed to regulate proton transfer by "shutting off" the protonic connectivity through the K-pathway after initiation of the O(2) reduction chemistry. This "shutoff" prevents a short-circuit of the proton-pumping machinery of the enzyme during the subsequent reaction steps.

Amino Acid Substitution↗
PubMed1998

The generation of proton electrochemical potential gradient by cytochrome c oxidase.

Cytochrome c oxidase, the terminal oxidase of mitochondria and some bacteria, catalyzes the four electron reduction of oxygen, and generates a proton electrochemical potential gradient (Delta microH). The recently determined structures of the bacterial and the bovine enzymes, together with studies of site directed mutants of a bacterial cytochrome c oxidase and a closely related ubiquinol oxidase, have greatly advanced our understanding of the mechanism by which oxygen reduction is coupled to the generation of Delta microH. Two different mechanisms contribute to the generation of Delta microH: protons that are consumed by the reduction of oxygen, are taken exclusively from the mitochondrial matrix ('consumed' protons), while other protons are translocated by the enzyme across the membrane ('pumped' protons). It is suggested that both proton consumption and proton pumping are driven by the electrostatic charging of the enzyme reaction center by the reducing electrons. Proton consumption is suggested to result from the electrostatically driven ejection of hydroxyls into the matrix that is catalyzed by a tyrosine residue in the reaction center. Proton pumping is suggested to result from the electrostatically driven translocation of a glutamate residue near the reaction center, and is assisted by secondary acceptors that release the translocated protons.

Animals↗
PubMed1990

Ferryl and hydroxy intermediates in the reaction of oxygen with reduced cytochrome c oxidase.

Cytochrome c oxidase catalyses the 4-electron reduction of dioxygen to water and translocates protons vectorially across the inner mitochondrial membrane. Proposed reaction pathways for the catalytic cycle of the O2 reduction are difficult to verify without knowing the structures of the intermediates, but we now have such information for the catalytic intermediates in the first steps of the reaction of O2 with cytochrome c oxidase from resonance Raman spectroscopy, a technique that enables iron-ligand stretching modes to be identified. Here we report on two more key intermediates: a ferryl-oxo (Fe4 = O2-) and a ferric-hydroxy (Fe3+--OH-) intermediate at the level of 3- and 4-electron reduction, respectively. We identified these intermediates by their characteristic iron-oxygen stretching frequencies (786 cm-1 for Fe4+ = O2-, and 450 cm-1 for Fe3+ -- OH-) and oxygen and deuterium isotope shifts. The oxo atom in the ferryl intermediate is hydrogen-bonded and the iron-oxygen bond in the hydroxy intermediate is anomalously weak. With the identification of the primary, ferryl and hydroxy intermediates, the predominant structures at almost all stages of O2 reduction are now known and the catalytic pathway can be described with more certainty.

Deuterium↗
PubMed2006

Nuclear respiratory factor 2 senses changing cellular energy demands and its silencing down-regulates cytochrome oxidase and other target gene mRNAs.

Cytochrome c oxidase (COX), the terminal enzyme of the electron transport chain, is a bigenomic enzyme with 13 subunits. The mechanism coordinating the transcription of these subunits is poorly understood. We investigated the role of nuclear respiratory factor-2 (NRF-2) in intragenomic regulation of nuclear COX genes. Vector-mediated short-hairpin RNA interference against NRF-2alpha reduced all 10 COX nuclear subunit mRNAs and mRNAs of other genes involved in mitochondrial function/biogenesis. NRF-2 binding site was necessary for the rat COX 4i1 promoter to down-regulate in response to decreased energy demands in primary neurons. Over-expression of NRF-2 protein prevented the down-regulation of transcriptional activity by TTX. Finally, NRF-2 binding sites in isolation were sufficient for modulating COX subunit 4i1 and 6A1 promoters' activity in response to decreased energy demand. These results indicate that NRF-2 is a vital part of a molecular mechanism that senses upstream energy signals and modulates COX transcriptional levels in mammalian cells.

Animals↗
PubMed1992

Laser flash photolysis studies of electron transfer mechanisms in cytochromes: an aromatic residue at position 82 is not required for cytochrome c reduction by flavin semiquinones or electron transfer from cytochrome c to cytochrome oxidase.

The influence of an aromatic side chain at position 82 of yeast iso-1-cytochrome c on the kinetics of its electron transfer reactions has been investigated using laser flash photolysis methods to compare a series of site-specific mutant cytochromes in their reduction by free flavin semiquinone and in electron transfer from reduced cytochrome to bovine cytochrome c oxidase. Although small (approximately 10%) but significant differences are observed between some of the mutants (S82, Y82, I82) and wild-type (F82) or G82 cytochrome in the second-order rate constant for reduction by lumiflavin semiquinone, these do not correlate with side-chain aromaticity. In the reaction between the ferrocytochromes and cytochrome c oxidase, significantly larger deviations from exponentiality are found for those mutants having aliphatic residues at position 82 than for wild type or Y82. We interpret the nonexponential behavior in terms of multiple orientations of the cytochromes within the oxidase binding site; the extent to which this occurs is apparently influenced by the character of the residue at position 82. However, a comparison of the average rate constants for electron transfer to cytochrome oxidase for the various mutants reveals that all are closely comparable to WT, except for I82 which is significantly slower (approximately threefold). These results, combined with those obtained previously from steady-state kinetic and thermodynamic measurements, suggest that the observed differences among the mutants are due to alterations in the mode of binding of the cytochrome to the oxidase, rather than to a specific requirement for the presence of an aromatic group at position 82.

Animals↗
PubMedDate not supplied

Differences in kinetic properties of cytochrome oxidase in mitochondria from rat tissues. A comparative study.

Substrate kinetic properties of cytochrome oxidase in rat liver, kidney, brain and heart mitochondria were examined using ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) as the electron donor system. Analysis of the substrate kinetics data revealed tissue-specific expression of kinetic components exhibiting differences with respect to Km, Vmax and Kcat/Km values. Regression analysis data suggest that the enzyme activity may be regulated in a tissue-specific manner.

Animals↗
PubMed2005

Proton exit channels in bovine cytochrome c oxidase.

Cytochrome c oxidase (CcO) is the terminal transmembrane enzyme of the respiratory electron transport chain in aerobic cells. It catalyzes the reduction of oxygen to water and utilizes the free energy of the reduction reaction for proton pumping, a process which results in a membrane electrochemical proton gradient. Although the structure of the enzyme has been solved for several organisms, the molecular mechanism of proton pumping and proton exit pathways remain unknown. In our previous work, the continuum electrostatic calculations were employed to evaluate the electrostatic potential, energies, and protonation state of bovine cytochrome c oxidase for different redox states of the enzyme. A possible mechanism of oxygen reduction and proton pumping via His291 was proposed. In this paper, using electrostatic calculations, we examine the proton exit pathways in the enzyme. By monitoring the changes of the protonation states, proton affinities, and energies of electrostatic interactions between the titratable groups in different redox states of CcO, we identified the clusters of strongly interacting residues. Using these data, we detected four possible proton exit points on the periplasmic side of the membrane (Lys171B/Asp173B, His24B/Asp25B, Asp51, and Asp300). We then were able to trace the proton exit pathways and to evaluate the energy profiles along the paths. On the basis of energetic considerations and the conservation of the residues in a protein sequence, the most likely exit pathway is one via the Lys171B/Asp173B site. The obtained results are fully consistent with our His291 model of proton pumping, and provide a rationale for the absence of proton leaking in CcO between the pumping strokes.

Animals↗
PubMed2004

Electrostatic study of the proton pumping mechanism in bovine heart cytochrome C oxidase.

Cytochrome c oxidase (CcO) is the terminal enzyme of the cell respiratory chain in mitochondria and aerobic bacteria. It catalyzes the reduction of oxygen to water and utilizes the free energy of the reduction reaction for proton pumping across the inner-mitochondrial membrane, a process that results in a membrane electrochemical proton gradient. Although the structure of the enzyme has been solved for several organisms, the molecular mechanism of proton pumping remains unknown. In the present paper, continuum electrostatic calculations were employed to evaluate the electrostatic potential, energies, and protonation state of bovine heart cytochrome c oxidase for different redox states of the enzyme along its catalytic cycle. Three different computational models of the enzyme were employed to test the stability of the results. The energetics and pH dependence of the P-->F, F-->O, and O-->E steps of the cycle have been investigated. On the basis of electrostatic calculations, two possible schemes of redox-linked proton pumping are discussed. The first scheme involves His291 as a pump element, whereas the second scheme involves a group linked to propionate D of heme a(3). In both schemes, loading of the pump site is coupled to ET between the two hemes of the enzyme, while transfer of a chemical proton is accompanied by ejection of the pumped H(+). The two models, as well as the energetics results are compared with recent experimental kinetic data. The proton pumping across the membrane is an endergonic process, which requires a sufficient amount of energy to be provided by the chemical reaction in the active site. In our calculations, the conversion of OH(-) to H(2)O provides 520 meV of energy to displace pump protons from a loading site and overall about 635 meV for each electron passing through the system. Assuming that the two charges are translocated per electron against the membrane potential of 200 meV, the model predicts an overall efficiency of 63%.

Animals↗
PubMed2004

Time-resolved optical absorption studies of cytochrome oxidase dynamics.

Time-resolved spectroscopic studies in our laboratory of bovine heart cytochrome c oxidase dynamics are summarized. Intramolecular electron transfer was investigated upon photolysis of CO from the mixed-valence enzyme, by pulse radiolysis, and upon light-induced electron injection into the cytochrome c/cytochrome oxidase complex from a novel photoactivatable dye. The reduction of dioxygen to water was monitored by a gated multichannel analyzer using the CO flow-flash method or a synthetic caged dioxygen carrier. The pH dependence of the intermediate spectra suggests a mechanism of dioxygen reduction more complex than the conventional unidirectional sequential scheme. A branched model is proposed, in which one branch produces the P form and the other branch the F form. The rate of exchange between the two branches is pH-dependent. A cross-linked histidine-phenol was synthesized and characterized to explore the role of the cross-linked His-Tyr cofactor in the function of the enzyme. Time-resolved optical absorption spectra, EPR and FTIR spectra of the compound generated after UV photolysis indicated the presence of a radical residing primarily on the phenoxyl ring. The relevance of these results to cytochrome oxidase function is discussed.

Animals↗
PubMed1989

Brain cytochrome oxidase activity of synaptic and nonsynaptic mitochondria during aging.

The activity of cytochrome c oxidase was studied in aging brain on non-synaptic and intra-synaptic mitochondria from frontal cerebral cortex, hippocampus and striatum of 4, 8, 12, 16, 20 and 24 month-old Sprague-Dawley rats. Specific activities of cytochrome oxidase were significantly higher in light synaptic mitochondria than in non-synaptic or heavy ones at all the ages examined. However, enzyme activity in light mitochondria from cerebral cortex remains unchanged during aging, being increased in hippocampus and striatum. These results indicate that aging affected not only the various cerebral area (macroheterogeneity), but also the different mitochondrial populations (subcellular heterogeneity).

Aging↗
PubMed1977

Near-neighbor relationships of the subunits of cytochrome c oxidase.

Cytochrome c oxidase in detergent dispersion has been cross-linked with two reversible cross-linking agents, dithiobissuccinimidylpropionate (DSP) and dimethyl-3,3'-dithiobispropionimidate (DTBP), and the cross-linked products formed have been analyzed by two-dimensional gel electrophoresis. Under mild reaction conditions, several subunit pairs were seen including II and V, V and VII, IV and VI. With higher levels of DSP, larger aggregates were seen until a cross-linked product with an apparent molecular weight of 140 000 was the predominant band on gels. This is the smallest molecular weight aggregate to contain all seven subunits of the enzyme and most likely represents the "unit" or two heme and two copper containing complex of cytochrome c oxidase.

Animals↗
PubMed1985

An analysis of the cellular localization of cytochrome oxidase in the lateral geniculate nucleus of the adult cat.

The distribution of cytochrome oxidase (C.O.) was examined in the normal adult cat lateral geniculate nucleus at the cellular and electron-microscopic levels. The darker reactivity of the X- and/or Y-receptive laminae (A, A1, magnocellular lamina C [Cm], and medial interlaminar nucleus [MIN]) compared with the lightly reactive W-receptive parvicellular lamina C (Cp) indicates that there are pathway-specific histochemical differences in the visual system of the cat. At the cellular level, darkly reactive large cells in the lateral geniculate nucleus (LGN) closely resemble class 1, Y-cells, in relative size and distribution, thus indicating that C.O. histochemistry may be used as a functional marker for these cells. Perigeniculate neurons are also darkly reactive. Neuronal classes 2, 4, and 3 (presumed X-cells, W-cells, and/or interneurons) have moderate to lightly reactive perikarya. The darkly reactive neuronal classes tend to receive relatively stronger proximal excitatory synaptic input than do the less reactive neuronal classes. Since all neuronal classes appeared to have darkly (or moderately) reactive dendrites, C.O. reactivity must differ between dendrite and soma of some neuronal classes. At the electron-microscopic level, distinct components of the neuropil tend to have specific levels of C.O. reactivity. The predominance of darkly reactive mitochondria in dendrites indicates that dendrites are metabolically very active. RLD and may F's, but few large axon terminals with round vesicles (RL) or small axon terminals with round vesicles (RS) profiles are darkly reactive, implying that specific classes of presynaptic structures are more active than others. Thus C.O. histochemistry may be useful for distinguishing not only functionally active neuronal classes such as Y-cells and perigeniculate (PG) neurons from less active neuronal classes, but also functionally more or less active parts of the same neuron including its dendrites, axons, and/or axon terminals.

Animals↗
PubMed1986

The localization of cytochrome oxidase in the LGN and striate cortex of postnatal kittens.

The distribution of cytochrome oxidase (C.O.) was examined in the lateral geniculate nucleus of the kitten during the first postnatal month and compared with the adult pattern. During the first week, most of the C.O. was localized within the perikarya of geniculate neurons. Perigeniculate neurons had darkly reactive dendrites as well as perikaya. A population of relatively large, darkly reactive neurons became distinguishable around the end of the first week, as the level of reactivity diminished to moderate-to-light within most medium and small neurons. On the basis of their relative size and pattern of distribution, most of the darkly reactive neurons are likely to represent ones that will later have class 1 morphology and develop Y receptive field properties. These cells normally undergo rapid growth earlier, and their growth is more adversely affected by early short-term monocular suture than other classes of less reactive geniculate neurons. Thus, in the LGN of developing kitten, C.O. histochemistry may be used as a functional marker for future class 1 Y-cells. The reactivity of the neuropil gradually increases as synapses with dendrites mature. At the electronmicroscopic level the increased reactivity of the neuropil is due mainly to an increase in the number of reactive mitochondria localized within the growing dendrites. In the developing striate cortex of postnatal kittens dark reactivity is localized in the outer part of layer II for the first 2 weeks and then disappears. Dark reactivity gradually increases in layer IV after the third week. The changes in C.O. reactivity accompany pathway-specific physiological and anatomical changes that occur during early postnatal development.

Animals↗
PubMed1987

Dependence of cytochrome oxidase activity in the rat lateral geniculate nucleus on retinal innervation.

Patterns of cytochrome oxidase (CO) activity were examined histochemically in the dorsal lateral geniculate nucleus (LGNd) and retina of pigmented rats. CO staining was not uniform and was distributed in a pattern similar to that of retinal afferents. Portions of the LGNd receiving an exclusively crossed projection were moderately reactive whereas regions receiving an uncrossed or overlapping crossed and uncrossed projection were darkly reactive. The dependence of oxidative metabolic activity in the LGNd on retinal innervation was verified in animals with unilateral enucleation. In adults, chronic monocular enucleation led to a decrease in CO staining in portions of the LGNd deprived of retinal input; in animals enucleated at birth, normal patterns of CO reactivity failed to develop and both LGNds had a more uniform pattern of moderate CO staining. Most neurons in the ganglion cell layer of the retina were moderately reactive for CO. However, there were approximately 3,000 darkly reactive cells, most of which appear to be ganglion cells. The darkly reactive cells were more numerous in the peripheral temporal retina. The laminar pattern of CO staining in the retina was similar to that described previously for carnivores and primates. The most reactive laminae were the inner and outer plexiform layers and the photoreceptor inner segments. Within the inner plexiform layer, sublamina a was more darkly stained than sublamina b. These results suggest that the physiological properties of crossed and uncrossed visual pathways in rats are functionally dissimilar at the level of both the retina and the LGNd.

Age Factors↗
PubMed1998

Phylogenetic relationships among onychophora from Australasia inferred from the mitochondrial cytochrome oxidase subunit I gene.

Nucleotide sequence variation in a region of the mitochondrial cytochrome oxidase subunit I (COI) gene (456 bp) was examined for 26 onychophorans representing 15 genera of the family Peripatopsidae from Australasia. Sequence analysis revealed high intergeneric COI sequence divergence (up to 20.6% corrected) but low amino acid substitution rates, with high levels of transitional saturation evident. Among unambiguously alignable sequences, parsimony and distance analyses revealed a broadly congruent tree topology, robust to various algorithms and statistical analysis. There are two major groupings. One, largely unresolved, consists entirely of Australian mainland taxa. The other, for which there is convincing support, includes all of the New Zealand and Tasmanian taxa together with one mainland Australian species. In respect of the two major groupings, this topology is consistent with previous morphologically based phylogenies and provides further evidence for an ancient radiation within the mainland Australian Onychophora. The biogeographic implications of the close affinities revealed between the Tasmanian and New Zealand taxa are discussed.

Animals↗
PubMed1983

Purification of Pseudomonas cytochrome oxidase (or nitrite reductase) by immunological methods.

A new purification procedure for the cytochrome oxidase from Pseudomonas aeruginosa based on immunoaffinity chromatography has been compared with the biochemical method and shown to be (i) fully competitive in terms of chemical homogeneity and enzymatic properties of the purified protein (ii) slightly less efficient in terms of total recovery and (iii) much more convenient in terms of the time required. A further evolution of the method that minimizes the number of purification steps and any stress to the native structure of the protein is suggested.

Amino Acids↗
PubMed1990

Effect of pH on CO recombination to cytochrome oxidase in intact mitochondria.

The rate of recombination of CO with fully reduced cytochrome oxidase in intact beef heart mitochondria was measured after flash photolysis at temperatures between 180 and 230K. At pH 7.4 a single Arrhenius slope corresponds to an apparent energy of activation (Ea) of 10.5 kcal/mol; the rate constants in 100% CO are twice those in the presence of 1% CO. At pH 5.5 with 100% CO, Ea's of 11.3 and 7.1 kcal/mol are observed above and below 210K, respectively, while Ea's of 7.4 and 11.1 kcal/mol are observed with 1% CO above and below 210K. At pH 9.0 Ea's of 9.2 (above 200K), 12.5 (190-200K), and 2.3 (below 190K) kcal/mol are observed with 1% CO; Ea's of 9.4, 13.4, and 2.4 kcal/mol are observed in the same temperature ranges with 100% CO present. The findings support a model with up to 4 energy barriers separating the heme region from the bulk medium with intermediate regions that can hold 1 or 2 CO, depending on pH.

Animals↗
PubMed1992

Phylogenetic relationships in the honeybee (genus Apis) as determined by the sequence of the cytochrome oxidase II region of mitochondrial DNA.

The complete nucleotide sequence of the mitochondrial cytochrome oxidase II (COII) gene was determined for five species of the honeybee (Genus: Apis): A. andreniformis, A. cerana, A. dorsata, A. florea, and A. koschevnikovi; these were then compared to the known sequence of the A. millifera gene from Crozier et al. (1989, Mol. Biol. Evol., 6: 399-411) and the wasp Excristes roborator (Liu and Beckenbach, 1992, Mol. Phylogenet. Evol., 1:41-52). Phylogenetic relationships were derived using the parasimony methods DNAPARS and PROTPARS of Felsenstein ("PHYLIP Manual Version 3.4, "University Herbarium, Univ. of California, Berkeley). The results suggest that A. dorsata is the most ancestral species, followed by the branching of A. florea/A. andreniformis and A. koschevnikovi, and then A. mellifera and A. cerana. This inference differs from the currently accepted view that considers the A. florea/A. andreniformis line to be the most ancestral.

Amino Acid Sequence↗