Palmitate decreases proton pumping of liver-type cytochrome c oxidase.

The H+/e- stoichiometry of reconstituted cytochrome c oxidase from bovine kidney, containing subunit VIaL (liver type), is 0.5 under standard conditions but 1.0 on addition of 1% cardiolipin to the lipid mixture (asolectin). Low concentrations of palmitate (half-maximal effect at 0.5 microm), but not laurate, myristate, stearate, oleate, 1-hexadecanol, palmitoyl glycerol and palmitoyl CoA, decreased the H+/e- ratio in the presence of cardiolipin from 1.0 to 0.5, accompanied by an increase of coupled, but not of uncoupled respiration of proteoliposomes. Cardiolipin and palmitate did not influence the H+/e- stoichiometry and respiration of reconstituted cytochrome c oxidase from bovine heart, containing subunit VIaH (heart-type). The H+/e- stoichiometry of the heart enzyme, however, is decreased from 1.0 to 0.5 by 5 mm intraliposomal ATP (instead of 5 mm ADP). It is assumed that palmitate binds to subunit VIaL. The partial uncoupling of proton pumping in cytochrome c oxidase is suggested to participate in mammalian thermogenesis.     

Regulation of Energy Transduction and Electron Transfer in Cytochrome c Oxidase by Adenine Nucleotides

Cytochrome c oxidase from bovine heart contains seven high-affinity binding sites for ATP or ADP and three additional only for ADP. One binding site for ATP or ADP, located at the matrix-oriented domain of the heart-type subunit VIaH, increases the H⁺/e^– stoichiometry of the enzyme from heart or skeletal muscle from 0.5 to 1.0 when bound ATP is exchanged by ADP. Two further binding sites for ATP or ADP, located at the cytosolic and the matrix domain of subunit IV, increases the K _M for Cytochrome c and inhibit the respiratory activity at high ATP/ADP ratios, respectively. We propose that thermogenesis in mammals is related to subunit VIaL of cytochrome c oxidase with a H⁺/e^– stoichiometry of 0.5 compared to 1.0 in the enzyme from bacteria or ectotherm animals. This hypothesis is supported by the lack of subunit VIa isoforms in cytochrome c oxidase from fish.     

Evolutionary aspects of cytochromec oxidase

The presence of additional subunits in cytochrome oxidase distinguish the multicellular eukaryotic enzyme from that of a simple unicellular bacterial enzyme. The number of these additional subunits increases with increasing evolutionary stage of the organism. Subunits I–III of the eukaryotic enzyme are related to the three bacterial subunits, and they are encoded on mito-chondrial DNA. The additional subunits are nuclear encoded. Experimental evidences are presented here to indicate that the lower enzymatic activity of the mammalian enzyme is due to the presence of nuclear-coded subunits. Dissociation of some of the nuclear-coded subunits (e.g., VIa) by laurylmaltoside and anions increased the activity of the rat liver enzyme to a value similar to that of the bacterial enzyme. Further, it is shown that the intraliposomal nucleotides influence the kinetics of ferrocytochromec oxidation by the reconstituted enzyme from bovine heart but not fromP. denitrificans. The regulatory function attributed to the nuclear-coded subunits of mammalian cytochromec oxidase is also demonstrated by the tissue-specific response of the reconstituted enzyme from bovine heart but not from bovine liver to intraliposomal ADP. These enzymes from bovine heart and liver differ in the amino acid sequences of subunits VIa, VIIa, and VIII. The results presented here are taken to indicate a regulation of cytochromec oxidase activity by nuclear-coded subunits which act like receptors for allosteric effectors and influence the catalytic activity of the core enzyme via conformational changes.     

Characterization of two different genes (cDNA) for cytochrome c oxidase subunit VIa from heart and liver of the rat.

By antibody screening of a rat liver and a rat heart cDNA library in lambda gt11 two clones coding for the liver- and heart-specific subunit VIa of rat cytochrome c oxidase were isolated. In the heart cDNA sequence a TAA stop codon was found in frame 18 bp 5' upstream of the first methionine codon, thus excluding a leader sequence for this protein. The two cDNAs contain the full-length coding region of two subunits. The amino acid sequences of the two subunits show only 50% homology, whereas 74% homology was found between rat heart and bovine heart subunit VIa. By Northern blot analysis it is shown that the gene for subunit VIa from heart is only expressed in heart and skeletal muscle, whereas that from liver is also expressed in kidney, brain, heart and weakly in muscle.     

Allosteric properties of cyanobacterial cytochrome c oxidase: inhibition of the coupled enzyme by ATP and stimulation by ADP

Thorough analysis of the cta operon of Synechocystis sp. PCC6803 (grown in high-concentration salt medium to enhance the expression of respiratory proteins) showed that, apart from ctaCDE and Fb genes potentially encoding subunits I, II, III, and a small pseudo-bacteria-like subunit-IV of unknown function, a large mitochondria-like cta-Fm gene and a pronounced terminator structure are additional components of the operon. The deduced cta Fm gene product shows approximately 50% and 20% sequence identity to the Saccharomyces cerevisiae and beef heart mitochondrial COIV proteins, respectively. It also shows amino acid regions (near the N terminus, on the cytosolic side) with conspicuous sequence similarities to adenylate-binding proteins such as ATP synthase beta subunit Walker A and B consensus regions or to adenylate kinase. We suggest that, similar to the situation with beef heart mitochondria, it is the mitochondria-like subunit-IV of the cyanobacterial aa3-type cytochrome-c oxidase that confers allosteric properties to the cyanobacterial enzyme, the H+/e- ratios of cytochrome c oxidation being significantly lowered by ATP (intravesicular or intraliposomal) but enhanced by ADP. Therefore, the antagonistic action of ATP and ADP was in a way that the redox reaction proper, was always significantly less affected than the coupled proton translocation. Evolutionary and ecological implications of the unusual allosteric regulation of a prokaryotic cytochrome-c oxidase is discussed.     

Tissue distribution of cytochrome c oxidase isoforms in mammals. Characterization with monoclonal and polyclonal antibodies

Monoclonal and polyclonal antibodies specific to the two isoforms of subunit VIa of bovine cytochrome c oxidase were generated and used to study the tissue distribution of this subunit pair in beef, human and rat. The so-called H-(heart) form was found exclusively in heart and skeletal muscle, whereas the so-called L-(liver) form was the only isoform present in brain, kidney, liver and smooth muscle. Little or no L-form was detected in skeletal muscle. In bovine heart no subunit VIa-L was detected, while in human heart the subunit VIa-H and VIa-L isoforms were present in roughly equal proportions. These results imply that, in humans, the deficiency of a subunit VIa isoform may have a different effect on the physiology of heart then on the physiology of skeletal muscle.     

Different isozymes of cytochrome c oxidase are expressed in bovine smooth muscle and skeletal or heart muscle

Cytochrome c oxidase (COX) was isolated from bovine smooth muscle (rumen), and compared with the enzyme from bovine liver, heart and skeletal muscle. A new isozyme of COX was found to be expressed in smooth muscle, which differs from the isozyme in liver and heart or skeletal muscle. SDS-PAGE as well as N-terminal amino acid sequencing of separated subunits from gel bands revealed the expression of the liver isoforms for subunits VIa and VIII and of the heart isoform for subunits VIIa in COX from smooth muscle.     

The cDNA for the heart/muscle isoform of bovine cytochrome c oxidase subunit VIa encodes a presequence.

We have used mixed oligonucleotide probes to isolate a cDNA for the heart/muscle isoform of cytochrome c oxidase (COX) subunit VIa (COX VIa-H) from a bovine heart cDNA library in lambda gt10. This cDNA, and a second one isolated upon rescreening, predict a 97 amino acid COX VIa precursor protein comprised of a 12 amino acid, basic presequence plus an 85 residue mature VIa protein. The presence of a presequence contrasts with the rat heart COX VIa cDNA.     

Isolation and properties of cytochrome c oxidase from rat liver and quantification of immunological differences between isozymes from various rat tissues with subunit-specific antisera

Cytochrome c oxidase was isolated from rat liver either by affinity chromatography on cytochrome-c--Sepharose 4B or by chromatography on DEAE-Sepharose. Dodecyl sulfate gel electrophoresis of both preparations showed the same subunit pattern consisting of 13 different polypeptides. Kinetic analysis of the two preparations gave a higher Vmax for the enzyme isolated by chromatography on DEAE-Sephacel. Specific antisera were raised in rabbits against nine of the ten nuclear endoded subunits. A monospecific reaction of each antiserum with its corresponding subunit was obtained by Western blot analysis, thus excluding artificial bands in the gel electrophoretic pattern of the isolated enzyme due to proteolysis, aggregation or conformational modification of subunits. With an antiserum against rat liver holocytochrome c oxidase a different reactivity was found by Western blot analysis for subunits VIa and VIII between isolated cytochrome c oxidases from pig liver or kidney and heart or skeletal muscle. For a quantitative analysis of immunological differences a nitrocellulose enzyme-linked immunosorbent assay was developed. Monospecific antisera against 12 of the 13 subunits of rat liver cytochrome c oxidase were titrated with increasing amounts of total mitochondrial proteins from different rat tissues dissolved in dodecyl sulfate and dotted on nitrocellulose. The absorbance of a soluble dye developed by the second peroxidase-conjugated antibody was measured. From the data the following conclusions were obtained: (a) The mitochondrial encoded catalytic subunits I-III of cytochrome c oxidase are probably identical in all rat tissues. (b) All nine investigated nuclear encoded subunits of cytochrome c oxidase showed immunological differences between two or more tissues. Large immunological differences were found between liver, kidney or brain and heart or skeletal muscle. Minor but significant differences were observed for some subunits between heart and skeletal muscle and between liver, kidney and brain. (c) Between corresponding nuclear encoded subunits of cytochrome c oxidase from fetal and adult tissues of liver, heart and skeletal muscle apparent immunological differences were observed. The data could explain cases of fatal infantile myopathy due to cytochrome c oxidase deficiency.     

Isoforms of cytochrome c oxidase in tissues and cell lines of the mouse.

The subunit pattern of immunopurified cytochrome c oxidase from cultured mouse cells and mature tissues of the mouse was investigated by electrophoretic analysis. In mature tissues two forms of cytochrome c oxidase could clearly be identified on the basis of differences in morbidity or staining intensity of subunits VIa and VIII. One form was present in muscle and heart, and the other in liver, kidney and spleen. In lung both forms were found. In the thymus, subunit VIII showed the characteristics of subunit VIII found in muscle and heart, whereas subunit VIa resembled subunit VIa found in liver. This suggest the existence of a third cytochrome c oxidase isoform. The subunits of cytochrome c oxidase from cultured cell lines showed no differences between the various cell lines and resembled those of mature mouse liver tissue. The cytochrome c oxidase isoform from cultured proliferating cells might therefore be the same as the one found in liver. Alternatively, it might represent either a normally occurring fetal isoform, or a form specific for poorly differentiated cultured cells.     

ADP increases the affinity for cytochrome c by interaction with the matrix side of bovine heart cytochrome c oxidase

The effect of intraliposomal ADP and ATP on the kinetics of cytochrome c oxidation in reconstituted bovine heart cytochrome c oxidase was measured by the photometric and polarographic method: 1. Intraliposomal ADP decreases and intraliposomal ATP increases the Km for cytochrome c when measured by the photometric assay under uncoupled conditions. 2. The above described effects are not obtained when the kinetics are measured with the polarographic assay. 3. Extraliposomal ATP increases the Km for cytochrome c similar to intraliposomal ATP, but this effect is measured with both methods of assay. 4. Under coupled conditions only a small decrease of the Km for cytochrome c by intraliposomal ADP is found.     

Regulation of respiration and energy transduction in cytochrome c oxidase isozymes by allosteric effectors

The binding of TNP-ATP (2 or 3-O-(2,4,6-trinitrophenyl)-ATP) to cytochrome c oxidase (COX) from bovine heart and liver and to the two-subunit COX of Paracoccus denitrificans was measured by its change of fluorescence. Three binding sites, two with high (dissociation constant K_d = 0.2 µM) and one with lower affinity (K_d = 0.9 µM), were found at COX from bovine heart and liver, while the Paracoccus enzyme showed only one binding site (K_d = 3.6 µM). The binding of [³⁵S]ATPaS was measured by equilibrium dialysis and revealed seven binding sites at the heart enzyme (K_d = 7.5 µM) and six at the liver enzyme (K_d = 12 µM). The Paracoccus enzyme had only one binding site (K_d = 16 µM). The effect of variable intraliposomal ATP/ADP ratios, but at constant total concentration of [ATP + ADP] = 5 mM, on the H⁺/e^- stoichiometry of reconstituted COX from bovine heart and liver were studied. Above 98% ATP the H⁺/e^- stoichiometry of the heart enzyme decreased to about half of the value measured at 100% ATP. In contrast, the H⁺/e^- stoichiometry of the liver enzyme was not influenced by the ATP/ADP ratio. It is suggested that high intramitochondrial ATP/ADP ratios, corresponding to low cellular work load, will decrease the efficiency of energy transduction and result in elevated thermogenesis for the maintenance of body temperature. (Mol Cell Biochem 174: 131–135, 1997)     

Control of mitochondrial membrane potential and ROS formation by reversible phosphorylation of cytochrome c oxidase

Phosphorylation of isolated cytochrome c oxidase from bovine kidney and heart, and of the reconstituted heart enzyme, with protein kinase A, cAMP and ATP turns on the allosteric ATP-inhibition at high ATP/ADP ratios. Also incubation of isolated bovine liver mitochondria only with cAMP andATP turns on, and subsequent incubation with Ca2+ turns off the allosteric ATP-inhibition of cytochrome c oxidase. In the bovine heart enzyme occur only three consensus sequences for cAMP-dependent phosphorylation (in subunits I, III and Vb). The evolutionary conservation of RRYS441 at the cytosolic side of subunit I, together with the above results, suggest that phosphorylation of Ser441 turns on the allosteric ATP-inhibition of cytochrome c oxidase. The results support the 'molecular-physiological hypothesis' [29], which proposes a low mitochondrial membrane potential through the allosteric ATP-inhibition. A hormone- or agonist-stimulated increase of cellular [Ca2+] is suggested to activate a mitochondrial protein phosphatase which dephosphorylates cytochrome c oxidase, turns off the allosteric ATP-inhibition and results in increase of mitochondrial membrane potential and ROS formation.     

Tissue-specificity overrides species-specificity in cytoplasmic cytochrome c oxidase polypeptides

With a high-resolving dodecyl sulfate electrophoretic system rat liver cytochrome c oxidase was separated into 13 different polypeptides. An antiserum against rat liver holocytochrome c oxidase immunoreacted with all 13 polypeptides, as demonstrated by immunofluorescence after transfer of the separated Coomassie blue-stained bands on nitrocellulose and coupling with FITC-protein A ("western blot"). Polypeptide-specific antisera reacted only with their corresponding polypeptides indicating that the various protein bands are represented by individual polypeptides. From total proteins of rat liver, kidney, heart, spleen and skeletal muscle mitochondria, only the cytochrome c oxidase polypeptides showed immunofluorescence with an antiserum against the rat liver holoenzyme. In contrast to the polypeptide from liver, polypeptide VIa from heart and skeletal muscle showed little or no reactivity, indicating a tissue-specificity of this polypeptide. Mitochondrial proteins from pig, bovine and blackbird heart were incubated with an antiserum against the rat liver holoenzyme. Immunoreaction was found with most cytochrome c oxidase polypeptides but not with polypeptide VIa. This result demonstrates less immunological relationship between tissue-specific polypeptides (VIa, VIIa and VIII) of the same species than between tissue-unspecific polypeptides of different species.     

Tissue- and species-specific expression of cytochrome c oxidase isozymes in vertebrates

Cytochrome c oxidase was isolated from brown fat tissue of the rat and compared with the isozymes from rat liver and heart, which differ at least in subunits VIa and VIII. ELISA titrations of COX from the three tissues with monospecific antisera to all 13 subunits of the rat liver enzyme showed differences between the three enzymes. The N-terminal amino-acid sequence analysis of subunits VIa and VIII from SDS-PAGE gel bands of the three enzymes indicates the occurrence of three different isozymes in the rat. N-terminal amino-acid sequence analysis of subunits VIa and VIII from cytochrome c oxidase of bovine and human heart demonstrates also species-specific differences in the expression of the 'liver-type' and 'heart-type' of subunits VIa and VIII.     

Control of mitochondrial membrane potential and ROS formation by reversible phosphorylation of cytochrome c oxidase

Phosphorylation of isolated cytochrome c oxidase from bovine kidney and heart, and of the reconstituted heart enzyme, with protein kinase A, cAMP and ATP turns on the allosteric ATP-inhibition at high ATP/ADP ratios. Also incubation of isolated bovine liver mitochondria only with cAMP and ATP turns on, and subsequent incubation with Ca²⁺ turns off the allosteric ATP-inhibition of cytochrome c oxidase. In the bovine heart enzyme occur only three consensus sequences for cAMP-dependent phosphorylation (in subunits I, III and Vb). The evolutionary conservation of RRYS⁴⁴¹ at the cytosolic side of subunit I, together with the above results, suggest that phosphorylation of Ser⁴⁴¹ turns on the allosteric ATP-inhibition of cytochrome c oxidase. The results support the 'molecular-physiological hypothesis' [29], which proposes a low mitochondrial membrane potential through the allosteric ATP-inhibition. A hormone- or agonist-stimulated increase of cellular [Ca²⁺]] is suggested to activate a mitochondrial protein phosphatase which dephosphorylates cytochrome c oxidase, turns off the allosteric ATP-inhibition and results in increase of mitochondrial membrane potential and ROS formation.