Interrelationships of copper, cytochrome oxidase, phospholipid synthesis and adenine nucleotide binding.

Studies are reported on the interrelationships in liver mitochondria of copper status, cytochrome oxidase activity, adenine nucleotide binding capacity and phospholipid synthesis. Direct exposure of mitochondria to cyanide or diethyldithiocarbamate depressed cytochrome oxidase activity; ADP-binding and phospholipid synthesis. Fractionation of mitochondria to increase the specific activity of cytochrome oxidase about 10-fold did not increase the affinity to bind ADP. Ageing of mitochondria or dialysis of mitochondria or mitochondrial membrane preparations against water or diethyldithiocarbamate at 0--2 degrees for 18 h did not decrease cytochrome oxidase activity or copper content of reisolated and resuspended mitochondria or mitochondrial membrane preparations, but considerably reduced the affinity to bind ADP. The respiratory inhibitors, fluoride and azide, at concentrations inhibitory to cytochrome oxidase did not reduce ADP-binding or phospholipid synthesis. Atractyloside did not inhibit cytochrome oxidase activity but did inhibit ADP-binding and phospholipid synthesis. Pre-incubation of mitochondrial membrane preparations with Cu++ increased the copper content and ADP-binding affinity. The results indicate that cytochrome oxidase is not the ADP-binding site of the mitochondrial membrane system and that reduced cytochrome oxidase activity per se does not depress binding affinity. Copper appears to be a component of the adenine nucleotide binding sites of mitochondrial membranes because the copper-complexing agents, cyanide and diethyldithiocarbamate, depressed ADP-binding, while increased mitochondrial membrane copper content increased ADP-binding.     

Mitochondrial copper metabolism and delivery to cytochrome c oxidase

Metals are essential elements of all living organisms. Among them, copper is required for a multiplicity of functions including mitochondrial oxidative phosphorylation and protection against oxidative stress. Here we will focus on describing the pathways involved in the delivery of copper to cytochrome c oxidase (COX), a mitochondrial metalloenzyme acting as the terminal enzyme of the mitochondrial respiratory chain. The catalytic core of COX is formed by three mitochondrially-encoded subunits and contains three copper atoms. Two copper atoms bound to subunit 2 constitute the Cu(A) site, the primary acceptor of electrons from ferrocytochrome c. The third copper, Cu(B), is associated with the high-spin heme a(3) group of subunit 1. Recent studies, mostly performed in the yeast Saccharomyces cerevisiae, have provided new clues about 1) the source of the copper used for COX metallation; 2) the roles of Sco1p and Cox11p, the proteins involved in the direct delivery of copper to the Cu(A) and Cu(B) sites, respectively; 3) the action mechanism of Cox17p, a copper chaperone that provides copper to Sco1p and Cox11p; 4) the existence of at least four Cox17p homologues carrying a similar twin CX(9)C domain suggestive of metal binding, Cox19p, Cox23p, Pet191p and Cmc1p, that could be part of the same pathway; and 5) the presence of a disulfide relay system in the intermembrane space of mitochondria that mediates import of proteins with conserved cysteines motifs such as the CX(9)C characteristic of Cox17p and its homologues. The different pathways are reviewed and discussed in the context of both mitochondrial COX assembly and copper homeostasis.     

Homocysteine Restricts Copper Availability Leading to Suppression of Cytochrome C Oxidase Activity in Phenylephrine-Treated Cardiomyocytes

Cardiomyocyte hypertrophy induced by phenylephrine (PE) is accompanied by suppression of cytochrome c oxidase (CCO) activity, and copper (Cu) supplementation restores CCO activity and reverses the hypertrophy. The present study was aimed to understand the mechanism of PE-induced decrease in CCO activity. Primary cultures of neonatal rat cardiomyocytes were treated with PE at a final concentration of l00 µM in cultures for 72 h to induce cell hypertrophy. The CCO activity was determined by enzymatic assay and changes in CCO subunit COX-IV as well as copper chaperones for CCO (COX17, SCO2, and COX11) were determined by Western blotting. PE treatment increased both intracellular and extracellular homocysteine concentrations and decreased intracellular Cu concentrations. Studies in vitro found that homocysteine and Cu form complexes. Inhibition of the intracellular homocysteine synthesis in the PE-treated cardiomyocytes prevented the increase in the extracellular homocysteine concentration, retained the intracellular Cu concentration, and preserved the CCO activity. PE treatment decreased protein concentrations of the COX-IV, and the Cu chaperones COX17, COX11, and SCO2. These PE effects were prevented by either inhibition of the intracellular homocysteine synthesis or Cu supplementation. Therefore, PE-induced elevation of homocysteine restricts Cu availability through its interaction with Cu and suppression of Cu chaperones, leading to the decrease in CCO enzyme activity.     

Disturbance of Copper Homeostasis Is a Mechanism for Homocysteine-Induced Vascular Endothelial Cell Injury

Elevation of serum homocysteine (Hcy) levels is a risk factor for cardiovascular diseases. Previous studies suggested that Hcy interferes with copper (Cu) metabolism in vascular endothelial cells. The present study was undertaken to test the hypothesis that Hcy-induced disturbance of Cu homeostasis leads to endothelial cell injury. Exposure of human umbilical vein endothelial cells (HUVECs) to concentrations of Hcy at 0.01, 0.1 or 1 mM resulted in a concentration-dependent decrease in cell viability and an increase in necrotic cell death. Pretreatment of the cells with a final concentration of 5 µM Cu in cultures prevented the effects of Hcy. Hcy decreased intracellular Cu concentrations. HPLC-ICP-MS analysis revealed that Hcy caused alterations in the distribution of intracellular Cu; more Cu was redistributed to low molecular weight fractions. ESI-Q-TOF detected the formation of Cu-Hcy complexes. Hcy also decreased the protein levels of Cu chaperone COX17, which was accompanied by a decrease in the activity of cytochrome c oxidase (CCO) and a collapse of mitochondrial membrane potential. These effects of Hcy were all preventable by Cu pretreatment. The study thus demonstrated that Hcy disturbs Cu homeostasis and limits the availability of Cu to critical molecules such as COX17 and CCO, leading to mitochondrial dysfunction and endothelial cell injury.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactylium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, and extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (holoenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (less than 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 micrometer, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 micrometer medium copper, holoenzyme secretion is maintained throughout cell growth. The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN(-)-insensitive, manganese form of this enzyme. Cells grown at 10 micrometer copper show 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

The human cytochrome c oxidase assembly factors SCO1 and SCO2 have regulatory roles in the maintenance of cellular copper homeostasis

Human SCO1 and SCO2 are metallochaperones that are essential for the assembly of the catalytic core of cytochrome c oxidase (COX). Here we show that they have additional, unexpected roles in cellular copper homeostasis. Mutations in either SCO result in a cellular copper deficiency that is both tissue and allele specific. This phenotype can be dissociated from the defects in COX assembly and is suppressed by overexpression of SCO2, but not SCO1. Overexpression of a SCO1 mutant in control cells in which wild-type SCO1 levels were reduced by shRNA recapitulates the copper-deficiency phenotype in SCO1 patient cells. The copper-deficiency phenotype reflects not a change in high-affinity copper uptake but rather a proportional increase in copper efflux. These results suggest a mitochondrial pathway for the regulation of cellular copper content that involves signaling through SCO1 and SCO2, perhaps by their thiol redox or metal-binding state.     

Cmc1p is a conserved mitochondrial twin CX9C protein involved in cytochrome c oxidase biogenesis

Copper is an essential cofactor of two mitochondrial enzymes: cytochrome c oxidase (COX) and Cu-Zn superoxide dismutase (Sod1p). Copper incorporation into these enzymes is facilitated by metallochaperone proteins which probably use copper from a mitochondrial matrix-localized pool. Here we describe a novel conserved mitochondrial metallochaperone-like protein, Cmc1p, whose function affects both COX and Sod1p. In Saccharomyces cerevisiae, Cmc1p localizes to the mitochondrial inner membrane facing the intermembrane space. Cmc1p is essential for full expression of COX and respiration, contains a twin CX9C domain conserved in other COX assembly copper chaperones, and has the ability to bind copper(I). Additionally, mutant cmc1 cells display increased mitochondrial Sod1p activity, while CMC1 overexpression results in decreased Sod1p activity. Our results suggest that Cmc1p could play a direct or indirect role in copper trafficking and distribution to COX and Sod1p.     

Crystal structure of human SCO1: implications for redox signaling by a mitochondrial cytochrome c oxidase "assembly" protein

Human SCO1 and SCO2 are copper-binding proteins involved in the assembly of mitochondrial cytochrome c oxidase (COX). We have determined the crystal structure of the conserved, intermembrane space core portion of apo-hSCO1 to 2.8 A. It is similar to redox active proteins, including thioredoxins (Trx) and peroxiredoxins (Prx), with putative copper-binding ligands located at the same positions as the conserved catalytic residues in Trx and Prx. SCO1 does not have disulfide isomerization or peroxidase activity, but both hSCO1 and a sco1 null in yeast show extreme sensitivity to hydrogen peroxide. Of the six missense mutations in SCO1 and SCO2 associated with fatal mitochondrial disorders, one lies in a highly conserved exposed surface away from the copper-binding region, suggesting that this region is involved in protein-protein interactions. These data suggests that SCO functions not as a COX copper chaperone, but rather as a mitochondrial redox signaling molecule.     

COX19 mediates the transduction of a mitochondrial redox signal from SCO1 that regulates ATP7A-mediated cellular copper efflux

SCO1 and SCO2 are metallochaperones whose principal function is to add two copper ions to the catalytic core of cytochrome c oxidase (COX). However, affected tissues of SCO1 and SCO2 patients exhibit a combined deficiency in COX activity and total copper content, suggesting additional roles for these proteins in the regulation of cellular copper homeostasis. Here we show that both the redox state of the copper-binding cysteines of SCO1 and the abundance of SCO2 correlate with cellular copper content and that these relationships are perturbed by mutations in SCO1 or SCO2, producing a state of apparent copper overload. The copper deficiency in SCO patient fibroblasts is rescued by knockdown of ATP7A, a trans-Golgi, copper-transporting ATPase that traffics to the plasma membrane during copper overload to promote efflux. To investigate how a signal from SCO1 could be relayed to ATP7A, we examined the abundance and subcellular distribution of several soluble COX assembly factors. We found that COX19 partitions between mitochondria and the cytosol in a copper-dependent manner and that its knockdown partially rescues the copper deficiency in patient cells. These results demonstrate that COX19 is necessary for the transduction of a SCO1-dependent mitochondrial redox signal that regulates ATP7A-mediated cellular copper efflux.     

Bioenergetic remodeling during cellular differentiation: changes in cytochrome c oxidase regulation do not affect the metabolic phenotype.

Myogenesis induces mitochondrial proliferation, a decrease in reactive oxygen species (ROS) production, and an increased reliance upon oxidative phosphorylation. While muscles typically possess 20%-40% excess capacity of cytochrome c oxidase (COX), undifferentiated myoblasts have only 5%-20% of the mitochondrial content of myotubes and muscles. We used two muscle lines (C2C12, Sol8) and 3T3-L1 pre-adipocytes to examine if changes in COX regulation or activity with differentiation cause a shift in metabolic phenotype (i.e., more oxidative, less glycolytic, less ROS). COX activity in vivo can be suppressed by its inhibitor, nitric oxide, or sub-optimal substrate (cytochrome c) concentrations. Inhibition of nitric oxide synthase via L-NAME had no effect on the respiration of adherent undifferentiated cells, although it did stimulate respiration of myoblasts in suspension. While cytochrome c content increased during differentiation, there was no correlation with respiratory rate or reliance on oxidative metabolism. There was no correlation between COX specific activity and oxidative metabolism between cell type or in relation to differentiation. These studies show that, despite the very low activities of COX, undifferentiated myoblasts and pre-adipocytes possess a reserve of COX capacity and changes in COX with differentiation do not trigger the shift in metabolic phenotype.     

Studies on the effects of copper deficiency on rat liver mitochondria. I. Changes in mitochondrial composition

As part of an investigation of the lesions of copper (Cu) deficiency a study was undertaken of the copper, iron, cytochrome and fatty acid composition of liver mitochondria from Cu deficient and Cu-adequate control rats. Cu concentrations were significantly decreased in whole liver, liver mitochondria and in blood plasma. Total iron was significantly increased in whole liver but remained at the normal level in mitochondria. Cytochrome c oxidase (EC 1.9.3.1) and its component cytochromes a and a3 were significantly reduced in liver mitochondria from Cu-deficient rats, whereas there was no effect on the concentration of cytochromes b, c1 and c. Evidence from comparisons between cytochrome c oxidase activity and the amount of enzyme present, as assessed from the mitochondrial cytochrome a and a3 content, suggests that in addition to an absolute loss of enzyme, Cu-deficiency adversely affects the efficiency of the residual enzyme. Severe Cu deficiency had no effect on 'ageing' or 'swelling' properties of liver mitochondria, indicating no marked effects on fatty acid composition. Fatty acid analyses demonstrated a slight but significant increase in docosapentenoic acid (22:5) of Cu-deficient mitochondria, but since this represents a minor component there was no change observed in the 'unsaturation index'. It was concluded that, in contrast to previous reports, Cu deficiency of the severity reported did not have a deleterious effect on the integrity and permeability of the inner mitochondrial membrane as exemplified by any qualitative modification of fatty acid constitution per se.     

Increased vulnerability of brain to estrogen withdrawal-induced mitochondrial dysfunction with aging

In the present study, to determine whether aging could increase the vulnerability of the brain to estrogen withdrawal-induced mitochondrial dysfunction, we measured the cytochrome c oxidase (COX) activity and mitochondrial adenosine triphosphate (ATP) content in hippocampi of 2 groups of ovariectomized (OVX) Wistar rats aged 2 months (young) and 9 months (middle-aged), respectively. In addition, effects of genistein and estradiol benzoate (EB) were tested also. We observed only a transient alteration of COX activity and mitochondrial ATP content in hippocampi of young OVX rats but a prolonged lowering of COX activity and mitochondrial ATP content in hippocampi of middle-aged OVX rats. This suggested that with aging compensatory mechanisms of mitochondrial function were attenuated, thus exacerbated estrogen withdrawal-induced mitochondrial dysfunction in hippocampi. Significantly, EB/genistein treatment reversed this estrogen withdrawal-induced mitochondrial dysfunction in both young and middle-aged rats suggesting that genistein may be used as a substitute for estradiol to prevent age-related disease such as Alzheimer’s disease in post-menopausal females.     

The utilization of copper and its role in the biosynthesis of copper-containing proteins in the fungus, Dactylium dendroides

Aspects of the utilization of copper by the fungus, Dactytium dendroides, have been studied. The organism grows normally at copper levels below 10 nM. Cells grown in medium containing 30 nM copper or less concentrate exogenous metal at all levels of added copper; copper uptake is essentially complete within 15 min and is not inhibited by cycloheximide, dinitrophenol or cyanide. These results indicate that copper absorption is not an energy-dependent process. The relationship between fungal copper status and the activities of three copper-containing enzymes, galactose oxidase, an extracellular enzyme, the cytosolic, Cu/Zn superoxide dismutase and cytochrome oxidase, has also been established. The synthesis of galactose oxidase protein (haloenzyme plus apo-enzyme) is independent of copper concentration. Cells grown in copper-free medium (< 10 nM copper) excrete normal amounts of galactose oxidase as an apoprotein. At medium copper levels below 5 μM, new cultures contain enough total copper to enable the limited number of cells to attain sufficient intracellular copper to support hologalactose oxidase production. As a result of cell division, however, the amount of copper available per cell drops to a threshold of approx. 10 ng/mg below which point only apogalactose oxidase is secreted. Above 5 μM medium copper, holoenzyme secretion is maintained throughout cell growth.The levels of the Cu/Zn superoxide dismutase respond differently in that the protein itself apparently is synthesized in only limited amounts in copper-depleted cells. Total cellular superoxide dismutase activity is maintained under such conditions by an increase in activity associated with the mitochondrial, CN^?-insensitive, manganese form of this enzyme. Cells grown at 10 μM copper shown 83% of their superoxide dismutase activity to be contributed by the Cu/Zn form compared to a 17% contribution to the total activity in cells grown at 30 nM copper, indicating that the biosynthesis of the Cu/Zn and Mn-containing enzymes is coordinated. The data show that the level of copper modulates the synthesis of the cytosolic superoxide dismutase. In contrast, the cytochrome oxidase activity of D. dendroides is independent of cellular copper levels obtainable. Thus, the data also suggest that these three enzymes utilize different cellular copper pools. As cells are depleted of copper by cell division, the available copper is used to maintain Cu/Zn superoxide dismutase and cytochrome oxidase activity; at very low levels of copper, only the latter activity is maintained. The induction of the manganisuperoxide dismutase in copper-depleted cells should have practical value in the isolation of this protein.     

Cytochrome c oxidase subassemblies in fibroblast cultures from patients carrying mutations in COX10, SCO1, or SURF1

Cytochrome c oxidase contains two redox-active copper centers (Cu(A) and Cu(B)) and two redox-active heme A moieties. Assembly of the enzyme relies on several assembly factors in addition to the constituent subunits and prosthetic groups. We studied fibroblast cultures from patients carrying mutations in the assembly factors COX10, SCO1, or SURF1. COX10 is involved in heme A biosynthesis. SCO1 is required for formation of the Cu(A) center. The function of SURF1 is unknown. Immunoblot analysis of native gels demonstrated severely decreased levels of holoenzyme in the patient cultures compared with controls. In addition, the blots revealed the presence of five subassemblies: three subassemblies involving the core subunit MTCO1 but apparently no other subunits; a subassembly containing subunits MTCO1, COX4, and COX5A; and a subassembly containing at least subunits MTCO1, MTCO2, MTCO3, COX4, and COX5A. As some of the subassemblies correspond to known assembly intermediates of human cytochrome c oxidase, we think that these subassemblies are probably assembly intermediates that accumulate in patient cells. The MTCO1.COX4.COX5A subassembly was not detected in COX10-deficient cells, which suggests that heme A incorporation into MTCO1 occurs prior to association of MTCO1 with COX4 and COX5A. SCO1-deficient cells contained accumulated levels of the MTCO1.COX4.COX5A subassembly, suggesting that MTCO2 associates with the MTCO1.COX4.COX5A subassembly after the Cu(A) center of MTCO2 is formed. Assembly in SURF1-deficient cells appears to stall at the same stage as in SCO1-deficient cells, pointing to a role for SURF1 in promoting the association of MTCO2 with the MTCO1.COX4.COX5A subassembly.     

Physical exercise regulates p53 activity targeting SCO2 and increases mitochondrial COX biogenesis in cardiac muscle with age

The purpose of this study was to outline the timelines of mitochondrial function, oxidative stress and cytochrome c oxidase complex (COX) biogenesis in cardiac muscle with age, and to evaluate whether and how these age-related changes were attenuated by exercise. ICR/CD-1 mice were treated with pifithrin-μ (PFTμ), sacrificed and studied at different ages; ICR/CD-1 mice at younger or older ages were randomized to endurance treadmill running and sedentary conditions. The results showed that mRNA expression of p53 and its protein levels in mitochondria increased with age in cardiac muscle, accompanied by increased mitochondrial oxidative stress, reduced expression of COX subunits and assembly proteins, and decreased expression of most markers in mitochondrial biogenesis. Most of these age-related changes including p53 activity targeting cytochrome oxidase deficient homolog 2 (SCO2), p53 translocation to mitochondria and COX biogenesis were attenuated by exercise in older mice. PFTμ, an inhibitor blocking p53 translocation to mitochondria, increased COX biogenesis in older mice, but not in young mice. Our data suggest that physical exercise attenuates age-related changes in mitochondrial COX biogenesis and p53 activity targeting SCO2 and mitochondria, and thereby induces antisenescent and protective effects in cardiac muscle.     

Characterization of YpmQ, an accessory protein required for the expression of cytochrome c oxidase in Bacillus subtilis

A search of the Bacillus subtilis genome identifies a potential homolog, ypmQ, of the inner mitochondrial membrane protein Sco1 from yeast. Sco1 has been found to aid the delivery of copper to cytochrome c oxidase. B. subtilis expresses two members of the cytochrome oxidase family, a cytochrome c oxidase that has two copper centers, Cu(A) and Cu(B), and a menaquinol oxidase that has only Cu(B). Deletion of ypmQ in B. subtilis depresses expression of cytochrome c oxidase but not menaquinol oxidase. Levels of cytochrome c oxidase recover when copper is added to the growth medium of the DeltaypmQ strain or when ypmQ is expressed from a plasmid. Neither treatment affects the amount or activity of menaquinol oxidase. YpmQ in which two conserved cysteines are replaced by serines and a conserved histidine is replaced by alanine do not complement the deletion of ypmQ even though these mutant forms are found in the membrane extract at a level similar to the wild type protein. We propose that the two cysteines and the histidine are critical for the function of YpmQ and suggest they are involved in copper exchange between YpmQ and the Cu(A) site of cytochrome c oxidase.