Copper deprivation potentiates oxidative stress in HL-60 cell mitochondria.

Cytochrome-c oxidase is the copper-dependent terminal respiratory complex (complex IV) of the mitochondrial electron transport chain whose activity in a variety of tissues is lowered by copper deficiency. Because inhibition of respiratory complexes increases the production of reactive oxygen species by mitochondria, it is possible that copper deficiency increases oxidative stress in mitochondria as a consequence of suppressed cytochrome-c oxidase activity. In this study, the activities of respiratory complex I + III, assayed as NADH:cytochrome-c reductase, complex II + III, assayed as succinate:cytochrome-c reductase, complex IV, assayed as cytochrome-c oxidase, and fumarase were measured in mitochondria from HL-60 cells that were grown for seven passages in serum-free medium that was either unsupplemented or supplemented with 50 n M CuSO4. Fumarase activity was not affected by copper supplementation, but the complex I + III:fumarase and complex IV:fumarase ratios were reduced 30% and 50%, respectively, in mitochondria from cells grown in the absence of supplemental copper. This indicates that copper deprivation suppressed the electron transfer activity of copper-independent complex I + III as well as copper-dependent complex IV. Manganese superoxide dismutase (MnSOD) content was also increased 49% overall in the cells grown in the absence of supplemental copper. Furthermore, protein carbonyl groups, indicative of oxidative modification, were present in 100-kDa and 90-kDa proteins of mitochondria from copper-deprived cells. These findings indicate that in cells grown under conditions of copper deprivation that suppress cytochrome-c oxidase activity, oxidative stress in mitochondria is increased sufficiently to induce MnSOD, potentiate protein oxidation, and possibly cause the oxidative inactivation of complex I.     

The preparative isolation of mitochondria from Chinese hamster ovary cells

A "hybrid" discontinuous gradient consisting of 6% Percoll overlaid on metrizamide separated mitochondria from other organelles in a Chinese hamster ovary cell postnuclear supernatant in a single 15-min centrifugation. The mitochondrial preparation contained about 25% of the mitochondrial marker, cytochrome-c oxidase, in a form that was about 90% latent. Based on the postnuclear supernatant, cytochrome-c oxidase activity was enriched approximately 45-fold. Trace amounts of lysosomal, rough endoplasmic reticular, Golgi, peroxisomal, plasma membrane, and cytosolic markers were found in the preparation. Electron microscopy revealed that the preparation consisted almost exclusively of mitochondria with only minor amounts of contaminating organelles. Analysis of the mitochondrial preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the mitochondrial preparation had a unique protein profile compared to the postnuclear supernatant and other gradient interfaces. Separation of the mitochondria into membrane and lumenal (matrix) fractions by treatment with 100 mM Na2CO3, pH 11.5, also indicated that the mitochondria were intact; they were rich in lumenal proteins. The data indicate that the mitochondria represent maximally about 2.2% of Chinese hamster ovary cell postnuclear supernatant protein. These isolated mitochondria should prove useful for problems in molecular cell biology.     

Nitrite biotransformation by mitochondria from the earthworm Eisenia foetida (Savigny).

1. Nitrite oxidase and cytochrome-c oxidase activity catalysed by cytochrome-aa3 were assayed in earthworms and rats. 2. Cytochrome-aa3 and intact mitochondria from the two species were anaerobically incubated in the presence of nitrite; the occurrence of mitochondria-induced nitrite biotransformations was evaluated by monitoring nitrite recovery in incubation medium. Possible nitric oxide production was also tested. 3. The ratio nitrite oxidase/cytochrome-c oxidase activity was much higher in earthworms than in rats. 4. Under anaerobic conditions and in the presence of respiratory substrates, earthworm mitochondria produced a time-dependent loss of nitrite in the incubation medium. On the contrary, rat mitochondria are unable to decrease environmental nitrite concentration. 5. Results support the notion that metabolic properties of earthworm mitochondria can be considered as an adaptation to chronic nitrite exposure, this toxicant being typically present in natural habitats of these worms.     

Characterization of cytochrome-c oxidase mutants in human fibroblasts

Skin fibroblasts were selected as having cytochrome-c oxidase deficiency by activity measurements in whole cells. Each cell line was cultured in sufficient amount to isolate mitochondria for biochemical characterization. Cytochrome-c oxidase was then examined by activity measurements, by heme determination and by polypeptide analysis using antibodies specific to the enzyme subunits. The cytochrome-c oxidase activity in the different cell lines ranged from 9% to 54% of that of normal fibroblasts. Heme determinations and polypeptide analysis established that the lowered cytochrome-c oxidase activity was due to reduced amounts of the complex in the mitochondrial inner membrane. In all cases, there was defective assembly of the enzyme, with the amounts of mitochondrially coded and nuclear coded subunits being reduced proportionally. These studies show that fibroblasts can be used for prenatal diagnosis of mitochondrial diseases and are a useful system in which to study mitochondrial biogenesis.     

The functional catalytic unit involved in proton pumping by rat liver cytochrome-c reductase and by cytochrome-c oxidase

The effect of partial inhibition on the protonmotive stoichiometry of cytochrome-c reductase and cytochrome-c oxidase in intact rat liver mitochondria was examined using myxothiazol and cyanide as inhibitors, respectively. No decrease in the stoichiometry of either enzyme was found. It is shown that this result is consistent with the individual electron transfer units in each case being fully coupled to proton translocation but not with pairs of electron transfer units working in concert in dimers.     

Optical and resonance Raman spectroscopy of the heme groups of the quinol-oxidizing cytochrome aa3 of Bacillus subtilis.

The cytochrome aa3-type terminal quinol oxidase of Bacillus subtilis catalyzes the four-electron reduction of dioxygen to water. It resembles the aa3-type cytochrome-c oxidase in using heme A as its active-site chromophores but lacks the CuA center and the cytochrome-c oxidizing activity of the mitochondrial enzyme. We have used optical and resonance Raman spectroscopies to study the B. subtilis oxidase in detail. The alpha-band absorption maximum of the reduced minus oxidized enzyme is shifted by 5-7 nm to the blue relative to most other aa3-type oxidases, and accordingly, we designate the Bacillus enzyme as cytochrome aa3-600. The shifted optical spectrum cannot be ascribed to an alteration in the strength of the hydrogen bond between the formyl group of the low-spin heme and its environment, as the Raman line assigned to this mode in aa3-600 has the same frequency and degree of resonance enhancement as the low-spin heme a formyl mode in most other aa3-type oxidases. Raman modes arise at 194 and 214 cm-1 in aa3-600, whereas a single band at about 214 cm-1 is assigned to the iron-histidine stretch for the other aa3-type oxidases. Possible explanations for the occurrence of these two modes are discussed. Comparison of formyl and vinyl modes and heme skeletal vibrational modes in different oxidation states of aa3-600 and of beef heart cytochrome-c oxidase shows a strong similarity, which suggests conservation of essential features of the heme environments in these oxidases.     

Structure and function of cytochrome-c oxidase

Recent works on the structure and the function of cytochrome-c oxidase are reviewed. The subunit composition of the mitochondrial enzyme depends on the species and is comprised of between 5 and 13 subunits. It is reduced to 1 to 3 subunits in prokaryotes. The complete amino acid composition has been derived from protein sequencing. Gene sequences are partially known in several eukaryote species. Metal centers are only located in subunits I and II. The mitochondrial cytochrome-c oxidase is Y-shaped; the arms of the Y cross the inner membrane, the stalk protrudes into the intermembrane space. The bacterial enzyme has a simpler, elongated shape. A number of data have been accumulated on the subunit topology and on their location within the protein. All available spectrometric techniques have been used to investigate the environment of the metal centers as well as their interactions. From the literature, attention must be paid to what may be considered or not as an active form. The steady improvement of the instrumentation has yielded evidence for different kinds of heterogeneities which could reflect the in vivo situation. The 'pulsed' and 'resting' conformers have been well characterized. The 'oxygenated' form has been identified as a peroxide derivative of the fully oxidized cytochrome-c oxidase. The mammalian enzyme has been isolated in fully active monomeric form which does not preclude the initially suggested dimeric behavior in situ. The role of the lipids is still largely investigated, mainly through reconstitution experiments. Kinetic studies of electron transfer between cytochrome c and cytochrome-c oxidase lead to a single catalytic site model to account for the multiphasic kinetics. Results related to the low temperature investigation of the intermediate steps in the reaction between oxygen and cytochrome-c oxidase received a sound confirmation by the resolution of compound A at room temperature. It is also pointed out that the so-called mixed valence state might not be a transient state in the catalytic reduction of oxygen. The functioning of cytochrome-c oxidase as a proton pump has been supported by a number of experimental results. Subunit III would be involved in this process. The redox link to the proton pump has been suggested to be at the Fea-CuA site. The molecular mechanism responsible for the proton pumping is still unknown.     

Isoforms of human cytochrome-c oxidase. Subunit composition and steady-state kinetic properties.

The subunit pattern and the steady-state kinetics of cytochrome-c oxidase from human heart, muscle, kidney and liver were investigated. Polyacrylamide gel electrophoresis of immunopurified cytochrome-c oxidase preparations suggest that isoforms of subunit VIa exist, which show differences in staining intensity and electrophoretic mobility. No differences in subunit pattern were observed between the other nucleus-encoded subunits of the various cytochrome-c oxidase preparations. Tissue homogenates, in which cytochrome-c oxidase was solubilised with laurylmaltoside, were directly used in the assays to study the cytochrome-c oxidase steady-state kinetics. Cytochrome-c oxidase concentrations were determined by immunopurification followed by separation and densitometric analysis of subunit IV. When studied in a medium of low ionic strength, the biphasic kinetics of the steady-state reaction between human ferrocytochrome c and the four human cytochrome-c oxidase preparations revealed large differences for the low-affinity TNmax (maximal turnover number) value, ranging from 77 s-1 for kidney to 273 s-1 for liver cytochrome-c oxidase at pH 7.4, I = 18 mM. It is proposed that the low-affinity kinetic phase reflects an internal electron-transfer step. For the steady-state reaction of human heart cytochrome-c oxidase with human cytochrome c, Km and TNmax values of 9 microM and 114 s-1 were found, respectively, at high ionic strength (I = 200 mM, pH 7.4). Only minor differences were observed in the steady-state activity of the various human cytochrome-c oxidases. The interaction between human cytochrome-c oxidase and human cytochrome-c proved to be highly specific. At high ionic strength, a large decrease in steady-state activity was observed when reduced horse, rat or bovine cytochrome c was used as substrate. Both the steady-state TNmax and Km parameters were strongly affected by the type of cytochrome c used. Our findings emphasize the importance of using human cytochrome c in kinetic assays performed with tissues from patients with a suspected cytochrome-c oxidase deficiency.     

Control processes in oxidative phosphorylation: kinetic constraints and stoichiometry

Control processes in oxidative phosphorylation have been studied in three experimental models. (1) In isolated yeast mitochondria, external ATP is a regulatory effector of cytochrome-c oxidase activity. In phosphorylating or uncoupling states, the relationships between respiratory rate and delta mu H+, and the respiratory rate and cytochrome-c oxidase reduction level are dependent on this kinetic regulation. (2) In rat liver mitochondria, the response of the respiratory rate to uncoupler addition is age-dependent: liver mitochondria isolated from young rats maintain a greater delta mu H+ than liver mitochondria isolated from adults, with the same respiratory rate obtained with the same concentration of uncoupler. This behaviour is linked to redox proton pump properties, i.e., to the degree of intrinsic uncoupling induced by uncoupler addition. (3) The effect of almitrine, a new kind of ATPase/ATPsynthase inhibitor, was studied in mammalian mitochondria. (i) Almitrine inhibits oligomycin-sensitive ATPase - it decreases the ATPase/O value without any change in delta mu H+; (ii) almitrine increased the mechanistic H+/ATP stoichiometry of ATPase/ATPsynthase; (iii) almitrine-induced changes in H+/ATPase stoichiometry depend on the flux magnitude through ATPase. These results are discussed in terms of the following interdependent parameters; flux value, force, pump efficiency and control coefficient.     

Assembly of cytochrome-c oxidase in the absence of assembly protein Surf1p leads to loss of the active site heme

Surf1p is a protein of the inner membrane of mitochondria that functions in the assembly of cytochrome-c oxidase. The specifics of the role of Surf1p have remained unresolved. Numerous mutations in human Surf1p lead to severe mitochondrial disease. A homolog of human Surf1p is encoded by the genome of the alpha-proteobacterium Rhodobacter sphaeroides, which synthesizes a mitochondrial-like aa(3)-type cytochrome-c oxidase. The gene for Surf1p was deleted from the genome of R. sphaeroides. The resulting aa(3)-type oxidase was purified and analyzed by biochemical methods plus optical and EPR spectroscopy. The oxidase that assembled in the absence of Surf1p was composed of three subpopulations with structurally distinct heme a(3)-Cu active sites. 50% of the oxidase lacked heme a(3), 10-15% contained heme a(3) but lacked Cu(BB), and 35-40% had a normal heme a(3) -Cu(B) active site with normal activity. Cu(A) assembly was unaffected. All of the oxidase contained low-spin heme a, but the environment of the heme a center was slightly altered in the 50% of the enzyme that lacked heme a(3). Introduction of a normal copy of the gene for Surf1p on an exogenous plasmid resulted in a single population of normally assembled, highly active enzyme. The data indicate that Surf1p plays a role in facilitating the insertion of heme a(3) into the active site of cytochrome-c oxidase. The results suggest that maturation of the heme a(3)-Cu(B) center is a step that limits the association of subunits I and II in the assembly of mitochondrial cytochrome oxidase.     

Purification of the Chlorella HUP1 hexose—proton symporter to homogeneity and its reconstitution in vitro

A prokaryotic biotin acceptor domain was fused to the carboxy terminal end of the Chlorella hexose—proton sym- porter. The plant symporter is biotinylated in vivo when expressed in Schizosaccharomyces pombe. The extended biotinylated transport protein is fully active, catalyzes accumulation of d -glucose analogs and restores growth of a glucose-uptake-deficient yeast strain. Crude membranes were solubilized with octyl-β-d -glucoside in the presence of Escherichia colil -α-phosphatidylethanolamine. Biotinylated symporter was purified to homogeneity by biotinavidin affinity chromatography. The symporter protein was reconstituted together with cytochrome-c oxidase prepared from beef heart mitochondria into proteo-liposomes. Cytochrome-c oxidase is a redox-driven H⁺-pump generating a proton motive force (inside negative and alkaline) while transferring electrons from cytochrome-c to oxygen; this energy is used by the symporter to accumulate d -glucose at least 30-fold. In the absence of the driving force the transport protein facilitates diffusion of d -glucose until the concentration equilibrium is reached. It was shown that maximal transport activity depends highly on the amount of co-reconstituted cytochrome-c oxidase and that the symporter possesses 10% of its in vivo turnover number under optimized in vitro transport conditions.     

Electron transfer between heme proteins and ceruloplasmin

Reduced cytochrome-c, reduced myoglobin and oxyhemoglobin respectively have been oxidized to oxidized cytochrome-c, metmyoglobin and methemoglobin by ceruloplasmin. Metmyoglobin and methemoglobin formation was stoichiometric while oxidized cytochrome-c reacted catalytically. Only 50% methemoglobin was formed which suggested that two hemes out of four could transfer electrons. Hydrogen peroxide was formed in the reaction of reduced cytochrome-c with ceruloplasmin.     

Influence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline modification on proton translocation and membrane potential of reconstituted cytochrome-c oxidase support "proton slippage".

Bovine heart cytochrome-c oxidase was reconstituted in liposomes and modified with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). EEDQ reacted mainly with subunits II and III and to a lower extent with subunit I, as shown by difference labeling with [14C]dicyclohexylcarbodiimide. EEDQ treatment of cytochrome-c oxidase vesicles influenced ferrocytochrome c-induced proton pumping by reducing maximally the H+/e- stoichiometry from 0.84 (control) to 0.24, but had only small effects on respiration, respiratory control ratio, and proton conductivity of the proteoliposomes. By titrating the reaction rate of the control and the modified cytochrome-c oxidase vesicles versus the membrane potential, as measured with a Ph3MeP+ electrode, saturation curves are obtained, which in both cases approach 225 mV. The ratios of electron transport rates of the two proton pumps at various membrane potentials decrease between 160 and 225 mV from about 2.2 to 1, indicating that the nonlinear flow/force relationship of these proton pumps is at least partly due to "slippage" of proton pumping.     

Modification of flow-force relationships by external ATP in yeast mitochondria

The purpose of this work is to measure protonmotive force and cytochrome reduction level under different respiratory steady states in isolated yeast mitochondria. The rate of respiration was varied by using three sets of conditions: (a) different external phosphate concentrations with a fixed concentration of ADP (ATP synthesis) and (b) different concentrations of carbonylcyanide m-chlorophenylhydrazone in the presence of oligomycin and carboxyatractylate (uncoupling) either in the absence or (c) in the presence of external ATP. ADP plus phosphate stimulates respiration more than uncoupler at the same protonmotive force value. However, the relationships between respiratory rate and protonmotive force were similar when stimulation was induced either by ADP + Pi or by carbonylcyanide m-chlorophenylhydrazone in the presence of ATP. At the same respiratory rate, cytochrome a + a3 is more reduced by uncoupler than by ADP + Pi additions. However, the relationships between respiratory rate and reduction level of cytochrome-c oxidase are similar both under ATP synthesis and with uncoupling conditions in the presence of external ATP. Control of respiration exerted by cytochrome-c oxidase, and support the view the condition mentioned above. This control was low when the respiratory rate was varied by the ATP synthesis rate; it increased as a function of the respiratory rate with uncoupler in the absence of ATP. ATP decreased this control under uncoupling conditions. These results suggest a regulatory effect of external ATP on cytochrome-c oxidase, and support the view that the relationships between respiratory rate and protonmotive force, on the one hand, and respiratory rate and the reduction level of cytochrome-c oxidase, on the other, depend respectively on the kinetic regulations of the system.     

Oxidized low-density lipoprotein and 15-deoxy-delta 12,14-PGJ2 increase mitochondrial complex I activity in endothelial cells

Oxidized lipids are capable of initiating diverse cellular responses through both receptor-mediated mechanisms and direct posttranslational modification of proteins. Typically, exposure of cells to low concentrations of oxidized lipids induces cytoprotective pathways, whereas high concentrations result in apoptosis. Interestingly, mitochondria can contribute to processes that result in either cytoprotection or cell death. The role of antioxidant defenses such as glutathione in adaptation to stress has been established, but the potential interaction with mitochondrial function is unknown and is examined in this article. Human umbilical vein endothelial cells (HUVEC) were exposed to oxidized LDL (oxLDL) or the electrophilic cyclopentenone 15-deoxy-Delta 12,14-PGJ2 (15d-PGJ2). We demonstrate that complex I activity, but not citrate synthase or cytochrome-c oxidase, is significantly induced by oxLDL and 15d-PGJ2. The mechanism is not clear at present but is independent of the induction of GSH, peroxisome proliferator-activated receptor (PPAR)-gamma, and PPAR-alpha. This response is dependent on the induction of oxidative stress in the cells because it can be prevented by nitric oxide, probucol, and the SOD mimetic manganese(III) tetrakis(4-benzoic acid) porphyrin chloride. This increased complex I activity appears to contribute to protection against apoptosis induced by 4-hydroxynonenal.     

Protein kinase C-epsilon coimmunoprecipitates with cytochrome oxidase subunit IV and is associated with improved cytochrome-c oxidase activity and cardioprotection

We have utilized an in situ rat coronary ligation model to establish a PKC-epsilon cytochrome oxidase subunit IV (COIV) coimmunoprecipitation in myocardium exposed to ischemic preconditioning (PC). Ischemia-reperfusion (I/R) damage and PC protection were confirmed using tetrazolium-based staining methods and serum levels of cardiac troponin I. Homogenates prepared from the regions at risk (RAR) and not at risk (RNAR) for I/R injury were fractionated into cell-soluble (S), 600 g low-speed centrifugation (L), Percoll/Optiprep density gradient-purified mitochondrial (M), and 100,000 g particulate (P) fractions. COIV immunoreactivity and cytochrome-c oxidase activity measurements estimated the percentages of cellular mitochondria in S, L, M, and P fractions to be 0, 55, 29, and 16%, respectively. We observed 18, 3, and 3% of PKC-delta, -epsilon, and -zeta isozymes in the M fraction under basal conditions. Following PC, we observed a 61% increase in PKC-epsilon levels in the RAR M fraction compared with the RNAR M fraction. In RAR mitochondria, we also observed a 2.8-fold increase in PKC-epsilon serine 729 phosphoimmunoreactivity (autophosphorylation), indicating the presence of activated PKC-epsilon in mitochondria following PC. PC administered before prolonged I/R induced a 1.9-fold increase in the coimmunoprecipitation of COIV, with anti-PKC-epsilon antisera and a twofold enhancement of cytochrome-c oxidase activity. Our results suggest that PKC-epsilon may interact with COIV as a component of the cardioprotection in PC. Induction of this interaction may provide a novel therapeutic target for protecting the heart from I/R damage.     

Regulation of Alternative Pathway Activity in Plant Mitochondria : Deviations from Q-Pool Behavior during Oxidation of NADH and Quinols

External NADH and succinate were oxidized at similar rates by soybean (Glycine max) cotyledon and leaf mitochondria when the cytochrome chain was operating, but the rate of NADH oxidation via the alternative oxidase was only half that of succinate. However, measurements of the redox poise of the endogenous quinone pool and reduction of added quinones revealed that external NADH reduced them to the same, or greater, extent than did succinate. A kinetic analysis of the relationship between alternative oxidase activity and the redox state of ubiquinone indicated that the degree of ubiquinone reduction during external NADH oxidation was sufficient to fully engage the alternative oxidase. Measurements of NADH oxidation in the presence of succinate showed that the two substrates competed for cytochrome chain activity but not for alternative oxidase activity. Both reduced Q-1 and duroquinone were readily oxidized by the cytochrome oxidase pathway but only slowly by the alternative oxidase pathway in soybean mitochondria. In mitochondria isolated from the thermogenic spadix of Philodendron selloum, on the other hand, quinol oxidation via the alternative oxidase was relatively rapid; in these mitochondria, external NADH was also oxidized readily by the alternative oxidase. Antibodies raised against alternative oxidase proteins from Sauromatum guttatum cross-reacted with proteins of similar molecular size from soybean mitochondria, indicating similarities between the two alternative oxidases. However, it appears that the organization of the respiratory chain in soybean is different, and we suggest that some segregation of electron transport chain components may exist in mitochondria from nonthermogenic plant tissues.     

The energy dependence of the chemical properties of cytochrome c oxidase

The addition of ATP to intact mitochondria induces a high- to low-spin state transition in a heme of oxidized cytochrome oxidase. This transition is dependent on the phosphate potential with one-half effect requiring a phosphate potential approximately 2 × 10³m⁻¹. The data are consistent with a phosphate potential dependent equilibrium between two oxidized forms of cytochrome oxidase (one oxidase per ATP). The addition of ATP to mitochondria decreases the rate of reaction of cyanide with oxidized cytochrome oxidase by at least 10³ and modifies both affinity and spectral change induced by adding cyanide to reduced cytochrome oxidase.     

Covalent and Noncovalent Dimers of the Cyanide-Resistant Alternative Oxidase Protein in Higher Plant Mitochondria and Their Relationship to Enzyme Activity

Evidence for a mixed population of covalently and noncovalently associated dimers of the cyanide-resistant alternative oxidase protein in plant mitochondria is presented. High molecular mass (oxidized) species of the alternative oxidase protein, having masses predicted for homodimers, appeared on immunoblots when the sulfhydryl reductant, dithiothreitol (DTT), was omitted from sodium dodecyl sulfate-polyacrylamide gel sample buffer. These oxidized species were observed in mitochondria from soybean (Glycine max [L.] Merr. cv Ransom), Sauromatum guttatum Schott, and mung bean (Vigna radiata [L.] R. Wilcz). Reduced species of the alternative oxidase were also present in the same mitochondrial samples. The reduced and oxidized species in isolated soybean cotyledon mitochondria could be interconverted by incubation with the sulfhydryl reagents DTT and azodicarboxylic acid bis(dimethylamide) (diamide). Treatment with chemical cross-linkers resulted in cross-linking of the reduced species, indicating a noncovalent dimeric association among the reduced alternative oxidase molecules. Alternative pathway activity of soybean mitochondria increased following reduction of the alternative oxidase protein with DTT and decreased following oxidation with diamide, indicating that electron flow through the alternative pathway is sensitive to the sulfhydryl/disulfide redox poise. In mitochondria from S. guttatum floral appendix tissue, the proportion of the reduced species increased as development progressed through thermogenesis.     

pH-induced alterations in the structure and activity of cytochrome c oxidase

The effects of pH on the activity and structure of beef heart cytochrome c oxidase have been studied in the pH range 5.0-7.6. (i) A group with pK of approximately 5.45 has been readily detected in the pH vs. activity curve. This group must be deprotonated to achieve maximal activity. (ii) A group with a similar pK (5.45) has been detected and contributes to the spectral character of the reduced oxidase. Over the range pH 5.0-7.6 no other acid-sensitive group contributes to the spectrum of the reduced oxidase. (iii) The oxidized oxidase shows at least three acid-sensitive groups contributing to the spectrum. One occurs in the pH 7 range and another in the pH 5.6 range; below pH 5.2 additional pH-sensitive groups are apparent. Accurate estimation of the pK's of the groups responsible for the spectral changes in the oxidized oxidase has not been possible. (iv) The spectrum of the "oxygenated" (428 nm) conformer of the oxidized protein is invariant over the range ph 5.5-7. (v) The changes occurring in the spectrum of the purified oxidase also occur in the protein contained in phospholipid vesicles. (vi) The data are discussed in terms of the mechanism by which the oxidase, during its in situ catalytic cycle, may give rise to the primary events in energy coupling.