Different reactivity of carboxylic groups of cytochrome c oxidase polypeptides from pig liver and heart

AbstractCytochrome c oxidase isolated from pig liver and heart was incubated with 1-ethyl-3-[3-(dimethylamino) propyl]carbodiimide and [14C]glycine ethyl ester in the presence and absence of cytochrome c. Labelling of individual subunits was determined after separation of the enzyme complexes into 13 polypeptides by SDS-gel electrophoresis. Polypeptide II and additional but different polypeptides were labelled in the liver and in the heart enzyme. Labelling of polypeptide II and of some other polypeptides could be partially or completely suppressed by cytochrome c. From the data two conclusions can be drawn: In addition to polypeptide II, other polypeptides take part in the binding of cytochrome c to cytochrome c oxidase; the binding domain for cytochrome c is different in pig liver and heart cytochrome c oxidase.Cytochrome c oxidase isozymeCytochrome c binding domain1-Ethyl-3-(3-dimethylaminopropyl)carbodiimideTissue specificit

The cDNA sequences of cytochrome c oxidase subunit VIa from carp and rainbow trout suggest the absence of isoforms in fishes1The sequence data in this paper have been submitted to the GenBank data library under the accession numbers: BankIt88656 U83980 (trout) and BankIt88644 U83907 (carp).1

AbstractThe cDNAs of subunit VIa of cytochrome c oxidase from rainbow trout liver and carp heart are presented, revealing 82% identity of their deduced amino acid sequences. The two cDNAs are evolutionary equally distant from the livertype (VIaL) and heart-type (VIaH) of mammalian subunit VIa. The data suggest that in ectotherm fishes no isoforms of subunit VIa occur, and that the postulated tissue-specific mechanism of thermogenesis in mammals, based on interaction of ATP with subunit VIaH (Frank, V. and Kadenbach, B. (1996) FEBS Lett. 382, 121–124), is absent.© 1997 Elsevier Science B.V. All rights reserved

Regulation of mitochondrial energy generation in health and disease

AbstractIn mammalian cytochrome c oxidase (COX) three of the ten nuclear coded subunits (VIa, VIIa, VIII) occur in tissue-specific isoforms. The isoform distribution, however, varies in liver and heart of different species. Subunit VIII is different in liver and heart of bovine, dog, rat and chicken, but identical in human (liver-type) on one hand, and sheep, rabbit and rainbow trout (heart-type) on the other hand, as determined by N-terminal sequencing. Two moles of trinitrophenyl-ATP bind to monomeric COX from bovine heart and one to COX from bovine liver with dissociation equilibrium constant (Kd) values of about 3 μM. One binding site at the heart enzyme is blocked by aa monoclonal antibody to subunit VIa-H. ATP (and/or ADP) interact with COX at two or three high-affinity binding sites, as shown by titration of the spectral changes of COX. Isolated COX from bovine heart was reconstituted with variable intraliposomal ATP/ADP ratios. By measuring the RCR (respiratory control ratio) and RCRVal (related to the valinomycin-respiration), which is a direct measure of the H+e−-stoichiometry (Wilson and Prochaska, Arch. Biochem. Biophys. 282 (1990) 413–420), almost complete inhibition of the proton pump activity of COX by high intraliposomal ATP concentrations was found. The vectorial uptake of protons for the formation of water, however, appears to be unaffected by nucleotides. This regulatory mechanism is assumed to have physiological significance for thermogenesis in muscle at rest. COX of fibroblasts from patients suffering from Leigh's syndrome, which is associated with a decreased COX activity, are suggested to have an incompletely assembled enzyme complex. This suggestion is further corroborated by the higher temperature-sensitivity of the enzyme when compared with COX from normal control fibroblasts. Defective regulation of COX via nuclear coded subunits is also proposed to cause mitochondrial diseases

Regulation of the H+/e− stoichiometry of cytochrome c oxidase from bovine heart by intramitochondrial ATP/ADP ratios

AbstractThis paper describes the effect of intramitochondrial ATP/ADP ratios on the H+/e− stoichiometry of reconstituted cytochrome c oxidase (COX) from bovine heart. At 100% intraliposomal ATP the H+/e− stoichiometry of the reconstituted enzyme is decreased to half of the value measured below 98% intraliposomal ATP (above 2% ADP), while it remains constant up to 100% ADP. The decrease is obtained with different COX preparations, independent of the absolute value of the H+/e− stoichiometry. Decrease of H+/e− stoichiometry is prevented by preincubation of the enzyme with a tissue-specific monoclonal antibody to subunit VIa-H (heart-type). Tissue-specific regulation of the efficiency of energy transduction in COX of muscle mitochondria could have a physilogical function in maintaining the body temperature at rest or sleep, i.e. at low ATP expenditure

Multiple Mechanisms Regulate Eukaryotic Cytochrome C Oxidase

Cytochrome c oxidase (COX), the rate-limiting enzyme of mitochondrial respiration, is regulated by various mechanisms. Its regulation by ATP (adenosine triphosphate) appears of particular importance, since it evolved early during evolution and is still found in cyanobacteria, but not in other bacteria. Therefore the “allosteric ATP inhibition of COX” is described here in more detail. Most regulatory properties of COX are related to “supernumerary” subunits, which are largely absent in bacterial COX. The “allosteric ATP inhibition of COX” was also recently described in intact isolated rat heart mitochondria

Stress-mediated generation of deleterious ROS in healthy individuals - role of cytochrome c oxidase

Psychosocial stress is known to cause an increased incidence of coronary heart disease. In addition, multiple other diseases like cancer and diabetes mellitus have been related to stress and are mainly based on excessive formation of reactive oxygen species (ROS) in mitochondria. The molecular interactions between stress and ROS, however, are still unknown. Here we describe the missing molecular link between stress and an increased cellular ROS, based on the regulation of cytochrome c oxidase (COX). In normal healthy cells, the 'allosteric ATP inhibition of COX' decreases the oxygen uptake of mitochondria at high ATP/ADP ratios and keeps the mitochondrial membrane potential (Δ

Biochemical analysis of fibroblasts from patients with cytochrome c oxidase-associated Leigh syndrome

AbstractCultured skin fibroblasts from four patients with Leigh syndrome and cytochrome c oxidase deficiency were studied. Mitochondrial DNA (mtDNA) analysis excluded large-scale deletions and known point mutations associated with Leigh syndrome. The COX activities were reduced to 18–44% of healthy probands, when measured in the presence of laurylmaltoside. COX activity from patients was shown to be more temperature sensitive than COX activity from control cells. In order to determine the subunit composition of COX immunoblotting studies were performed using mono- and polyclonal antibodies to distinct subunits. A monoclonal antibody to subunit IV crossreacted with two unknown proteins of higher apparent molecular weight in mitochondria from three patients, but not in mitochondria from control and the fourth patient. Quantification of immunoreactivity revealed a decrease of subunits II/III and IV parallel to the determined enzyme activity. In contrast, a variable amount of subunit VIIa (and/or VIIb) was found in mitochondria from different patients. The results indicate a defective COX holoenzyme complex in patients with Leigh syndrome and suggest different molecular origins of the defect