Selective removal of subunit VIb increases the activity of cytochrome c oxidase.

Bovine heart cytochrome c oxidase was gel-filtered on Sephacryl S-300 in 0.05% dodecyl maltoside and in the presence or absence of 1 M KCl. The presence of KCl selectively removed subunit VIb from the enzyme complex, resulting in about doubling of enzymatic activity and an increase of the Km for ferrocytochrome c. In contrast, the proton pumping activity of the enzyme was unchanged. The increase of activity is due to removal of subunit VIb and not of lipids, because titration with asolectin or dodecyl maltoside could not abolish the difference in activity between the 12- and 13-subunit enzyme. Attempts to reconstitute cytochrome c oxidase from its separated components were unsuccessful. It is concluded that subunit VIb suppresses the activity of the mammalian enzyme complex by interaction with the active center.     

Species-specific expression of cytochrome c oxidase subunit III mRNA

1. A cDNA probe encoding cytochrome c oxidase subunit III cloned from rat liver mitochondria was used to quantify mRNA levels in rat, mouse and rabbit tissues. This was compared to its phenotypic expression, using enzyme activity. 2. Enzyme activities were highest in mouse, intermediate in rat, and lowest in rabbit tissues. 3. Subunit III mRNA levels were easily quantified in rat, but could not be accurately measured in rabbit or mouse tissues despite high cytochrome c oxidase activities. 4. Significant subunit III sequence divergence must exist, among these species. Caution should be exercised in quantifying the expression of this mitochondrial gene.     

An active cytochrome c oxidase depleted of subunit III prepared by covalent chromatography on yeast cytochrome c

A covalent chromatography technique is described for the preparation of an active cytochrome c oxidase from bovine heart devoid of subunit III. Yeast cytochrome c is immobilized on a Sepharose 4B gel, its cysteine 107 activated and reacted with the oxidase. Elution with Triton X-100 releases an oxidase devoid of subunit III, which is recovered after elution with β-mercaptoethanol.     

Diphosphatidylglycerol reactivation of lipid-depleted beef heart cytochrome c oxidase: effect of subunit III removal

Beef heart cytochrome c oxidase can be fully delipidated by Triton X-100 using an alkaline pH, high ionic strength incubation followed by glycerol gradient centrifugation at pH 8 in 1% Triton X-100. Unfortunately, this procedure removes not only the 2-3 diphosphatidylglycerol (DPG) molecules that are tightly bound to each heme aa3 complex, but also removes subunit III. A one-to-one correlation exists between the percent of subunit III removed and the percent of tightly bound DPG extracted, suggesting a possible tight binding of these two molecules. However, based upon regeneration of most of the electron transport activity of the subunit III deficient complex by exogenous DPG, it appears that the functional DPG binding site must be located on a subunit other than subunit III.     

Lipid and subunit III depleted cytochrome c oxidase purified by horse cytochrome c affinity chromatography in lauryl maltoside

Cytochrome oxidase is purified from rat liver and beef heart by affinity chromatography on a matrix of horse cytochrome c-Sepharose 4B. The success of this procedure, which employs a matrix previously found ineffective with beef or yeast oxidase, is attributed to thorough dispersion of the enzyme with nonionic detergent and a low density of cross-linking between the lysine residues of cytochrome c and the cyanogen bromide activated Sepharose. Beef heart oxidase is purified in one step from mitochondrial membranes solubilized with lauryl maltoside, yielding an enzyme of purity comparable to that obtained on a yeast cytochrome c matrix [Azzi, A., Bill, K., & Broger, C. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2447-2450]. Rat liver oxidase is prepared by hydroxyapatite and horse cytochrome c affinity chromatography in lauryl maltoside, yielding enzyme of high purity (12.5-13.5 nmol of heme a/mg of protein), high activity (TN = 270-400 s-1), and very low lipid content (1 mol of DPG and 1 mol of PI per mol of aa3). The activity of the enzyme is characterized by two kinetic phases, and electron transfer can be stimulated to maximal rates as high as 650 s-1 when supplemented with asolectin vesicles. The rat liver oxidase purified by this method does not contain the polypeptide designated as subunit III. Comparisons of the kinetic behavior of the enzyme in intact membranes, solubilized membranes, and the purified delipidated form reveal complex changes in kinetic parameters accompanying the changes in state and assay conditions, but do not support previous suggestions that subunit III is a critical factor in the binding of cytochrome c at the high-affinity site on oxidase or that cardiolipin is essential for the low-affinity interaction of cytochrome c. The purified rat liver oxidase retains the ability to exhibit respiratory control when reconstituted into phospholipid vesicles, providing definitive evidence that subunit III is not solely responsible for the ability of cytochrome oxidase to produce or respond to a membrane potential or proton gradient.     

cAMP-dependent tyrosine phosphorylation of subunit I inhibits cytochrome c oxidase activity

Signaling pathways targeting mitochondria are poorly understood. We here examine phosphorylation by the cAMP-dependent pathway of subunits of cytochrome c oxidase (COX), the terminal enzyme of the electron transport chain. Using anti-phospho antibodies, we show that cow liver COX subunit I is tyrosinephosphorylated in the presence of theophylline, a phosphodiesterase inhibitor that creates high cAMP levels, but not in its absence. The site of phosphorylation, identified by mass spectrometry, is tyrosine 304 of COX catalytic subunit I. Subunit I phosphorylation leads to a decrease of V(max) and an increase of K(m) for cytochrome c and shifts the reaction kinetics from hyperbolic to sigmoidal such that COX is fully or strongly inhibited up to 10 mum cytochrome c substrate concentrations, even in the presence of allosteric activator ADP. To assess our findings with the isolated enzyme in a physiological context, we tested the starvation signal glucagon on human HepG2 cells and cow liver tissue. Glucagon leads to COX inactivation, an effect also observed after incubation with adenylyl cyclase activator forskolin. Thus, the glucagon receptor/G-protein/cAMP pathway regulates COX activity. At therapeutic concentrations used for asthma relief, theophylline causes lung COX inhibition and decreases cellular ATP levels, suggesting a mechanism for its clinical action.     

Monomerization of cytochrome oxidase may be essential for the removal of subunit III

1. Incubation of cytochrome oxidase, under conditions used as initial steps in treatment to remove subunit III, causes at least partial monomerization of the enzyme. 2. The extent of removal of subunit III by anion-exchange fast protein liquid chromatography (FPLC) is much increased if the enzyme is fully monomerized before it is applied to the column. 3. Subunit III is incompletely removed by chymotrypsin treatment. A digestion product of subunit III migrating in SDS-PAGE like subunit IV, is detected with specific antibodies. The amount of this product is reduced when monomerization is increased by raising the detergent/protein ratio. 4. The results suggest that monomerization facilitates removal of subunit III and exposes it to further chymotrypsin digestion. We propose that subunit III is at least in part located in the junction between the monomers in the cytochrome oxidase dimer.     

Cytochrome c oxidase depleted of subunit III: proton-pumping, respiratory control, and pH dependence of the midpoint potential of cytochrome a

An examination of respiratory control, proton pumping, and the pH dependence of the redox potential of cytochrome a is reported for subunit III-depleted rat liver cytochrome oxidase prepared by chromatography in laurylmaltoside. The results indicate a facilitating rather than essential role for subunit III in these properties related to energy conservation.     

Effect of oleic acid on mitochondrial cytochrome c oxidase activity

Both Km and Vmax values of cytochrome c oxidase for cytochrome c were elevated in oleic acid-incorporated mitochondria, whereas the amount of oleic acid incorporated into submitochondrial particles was smaller than that into mitochondria and the fatty acid had little effect on the enzyme activity. The degree of change in the bulk membrane fluidity was, however, almost the same in mitochondria and submitochondrial particles. Solubilized cytochrome c oxidase was insensitive to the effect of oleic acid. Oleic acid may act as a modifier of the interaction between cytochrome c oxidase and membrane lipids.     

Independent control of respiration in cytochrome c oxidase vesicles by pH and electrical gradients

The effects of altering the pH and electrical components of the membrane potential on the visible spectra and oxygen consumption rates of cytochrome oxidase vesicles were examined during steady-state respiration using cytochrome c as the substrate. Heme a was found to be 30-55% reduced in the presence of a membrane potential, becoming more reduced when the electrical gradient (delta psi) was abolished by valinomycin and more oxidized when the pH gradient (delta pH) was abolished by nigericin, with little increase (1.2-1.8-fold) in the rates of oxygen consumption in either case. When both gradients were eliminated, heme a reduction was close to initial levels, and activity was stimulated up to 8-fold. The magnitude of the changes in heme a reduction levels upon elimination of a gradient component was shown to be positively correlated with the magnitude of the respiratory control ratio of the vesicle preparation. Kinetic analysis of the dependence of oxidase activity on cytochrome c concentration indicated that changes in the Michaelis constant of the enzyme for its substrate are not a major factor in regulation by either delta pH or delta psi. These results suggest a dual mechanism for respiratory control in cytochrome oxidase vesicles under steady-state conditions, in which the electrical gradient predominantly affects electron transfer from cytochrome c to heme a, possibly by altering the reduction potential of heme a, while the pH gradient affects electron transfer from heme a (CuA) to heme a3 (CuB), possibly by a conformationally mediated change in the reduction potential of heme a3 or in the kinetics of the electron-transfer process.     

Cloning of Paracoccus cytochrome c oxidase subunit II

Cytochrome c oxidase from Paracoccus denitrificans is composed of two subunits, yet is active in both electron transport and proton translocation. A cloning approach and immunologic screening protocol is described for the isolation of the subunit II gene expressed in E. coli. DNA sequencing should establish the extent of homology to eukaryotic oxidase.     

A pathogenic 15-base pair deletion in mitochondrial DNA-encoded cytochrome c oxidase subunit III results in the absence of functional cytochrome c oxidase

A 15-base pair, in-frame, deletion (9480del15) in the mitochondrial DNA (mtDNA)-encoded cytochrome c oxidase subunit III (COX III) gene was identified previously in a patient with recurrent episodes of myoglobinuria and an isolated COX deficiency. Transmitochondrial cell lines harboring 0, 97, and 100% of the 9480del15 deletion were created by fusing human cells lacking mtDNA (rho(0) cells) with platelet and lymphocyte fractions isolated from the patient. The COX III gene mutation resulted in a severe respiratory chain defect in all mutant cell lines. Cells homoplasmic for the mutation had no detectable COX activity or respiratory ATP synthesis, and required uridine and pyruvate supplementation for growth, a phenotype similar to rho(0) cells. The cells with 97% mutated mtDNA exhibited severe reductions in both COX activity (6% of wild-type levels) and rates of ATP synthesis (9% of wild-type). The COX III polypeptide in the mutant cells, although translated at rates similar to wild-type, had reduced stability. There was no evidence for assembly of COX I, COX II, or COX III subunits in a multisubunit complex in cells homoplasmic for the mutation, thus indicating that there was no stable assembly of COX I with COX II in the absence of wild-type COX III. In contrast, the COX I and COX II subunits were assembled in cells with 97% mutated mtDNA.     

Effect of pH on the spectrum of cytochrome c oxidase hydrogen peroxide complex

Hydrogen peroxide binding to ferric cytochrome c oxidase in proteoliposomes brings about a red-shift of the enzyme Soret band and increased absorption in the visible range with two prominent peaks at approx. 570 and 607 nm. The molar absorptivity of the H2O2-induced difference spectrum is virtually pH-independent in the Soret band and at 570 nm, whereas the peak at 607 nm increases approx. 3-fold upon alkalinization in a narrow pH range 6.0-7.2, the effect being reversible. The pH profile of this transition indicates ionization of two acid-base groups with close pK values of 6.7. The lineshape of the peroxide compound difference spectrum is found to respond to pH changes inside the proteoliposomes. It is suggested that peroxide-complexed enzyme can undergo a pH-dependent transition to a form with increased extinction at 605-607 nm, possibly corresponding to the 420 nm (or 'pulsed') conformer of the ferric cytochrome oxidase formed as an early product of the enzyme oxidation. Accordingly, relaxation of the '420 nm' form to the resting state would be linked to an uptake of two protons from the M-aqueous phase. This protolytic reaction might be a partial step of the cytochrome oxidase proton pumping mechanism or it could serve to regulate interconversion between the active 'pulsed' and less active 'resting' states of the enzyme in the membrane.     

Homology between bacterial DNA and bovine mitochondrial DNA encoding cytochrome c oxidase subunit III

A segment of mitochondrial DNA encoding the bovine cytochrome c oxidase subunit III gene was isolated and inserted into an Escherichia coli plasmid vector. A 556 base pair fragment of the insert DNA representing about 70% of the 3'-end of the subunit III gene was used to search for homology with bacterial DNA from strains that contain heme aa3-type cytochrome c oxidases. Bacillus subtilis, Thermus thermophilus, and PS3 DNAs all showed strong hybridization to the probe, whereas Paracoccus denitrificans and Rhodopseudomonas sphaeroides DNAs showed only weak hybridization to the probe, even under low stringency conditions.     

Cyanide binding to bovine heart cytochrome c oxidase depleted of subunit III by treatment with lauryl maltoside

Subunit III was removed from beef heart cytochrome oxidase by incubation of the isolated enzyme at 25 degrees C for 24 h in lauryl maltoside buffer at a detergent to protein ratio of 10:1 (w:w). During the course of the incubation, the reaction of the enzyme with cyanide was followed by spectrophotometry in the Soret region. The starting material binds cyanide in a multiexponential process with 70% of the reaction occurring during the slow phase of the reaction at an observed rate of 3.85 X 10(-5) S-1 with 1 mM KCN. More of the enzyme binds cyanide during the fast phase of the reaction at an observed rate of 3.8 X 10(-3) S-1 as subunit III is removed by lauryl maltoside. After 24 h of incubation in lauryl maltoside, the enzyme reacts with cyanide completely in a rapid, single exponential process. When the protein from such an incubation is recovered by cytochrome c affinity chromatography and analyzed for its subunit content, subunit III is absent. The position of the Soret maximum of the oxidized enzyme shifts from its maximum at 418 nm in the starting material to 422 nm in the subunit III-depleted enzyme. The subunit III-depleted enzyme binds cyanide completely in a simple bimolecular reaction with a rate constant of 3.8 M-1 S-1. We discuss this result in terms of the possible structural and functional roles for subunit III in the cytochrome oxidase complex.     

Control of cytochrome c oxidase activity by nitric oxide

Over the past decade it was discovered that, over-and-above multiple regulatory functions, nitric oxide (NO) is responsible for the modulation of cell respiration by inhibiting cytochrome c oxidase (CcOX). As assessed at different integration levels (from the purified enzyme in detergent solution to intact cells), CcOX can react with NO following two alternative reaction pathways, both leading to an effective, fully reversible inhibition of respiration. A crucial finding is that the rate of electron flux through the respiratory chain controls the mechanism of inhibition by NO, leading to either a "nitrosyl" or a "nitrite" derivative. The two mechanisms can be discriminated on the basis of the differential photosensitivity of the inhibited state. Of relevance to cell pathophysiology, the pathway involving the nitrite derivative leads to oxidative degradation of NO, thereby protecting the cell from NO toxicity. The aim of this work is to review the information available on these two mechanisms of inhibition of respiration.