Differential loss of cytochrome-c oxidase subunits in ischemia-reperfusion injury: exacerbation of COI subunit loss by PKC-epsilon inhibition

We have previously described a PKC-epsilon interaction with cytochrome oxidase subunit IV (COIV) that correlates with enhanced CO activity and cardiac ischemic preconditioning (PC). We therefore investigated the effects of PC and ischemia-reperfusion (I/R) injury on CO subunit levels in an anesthetized rat coronary ligation model. Homogenates prepared from the left ventricular 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), gradient-purified mitochondrial (M), and 100,000 g particulate (P) fractions. In RAR tissue, PC (2 cycles of 5-min ischemia and 5-min reperfusion) decreased the COI in the P fraction ( approximately 29% of total cellular COI), suggesting changes in interfibrillar mitochondria. After 30 min of ischemia and 120 min of reperfusion, total COI levels decreased in the RAR by 72%. Subunit Va was also downregulated by 42% following prolonged I/R in the RAR. PC administered before I/R reduced the loss of COI in the M and P fractions approximately 30% and prevented COVa losses completely. We observed no losses in subunits Vb and VIIa following I/R alone; however, significant losses occurred when PC was administered before prolonged I/R. Delivery of a cell-permeable PKC-epsilon translocation inhibitor (epsilonV1-2) to isolated rat hearts before prolonged I/R dramatically increased COI loss, suggesting that PKC-epsilon protects COI levels. We propose that additional measures to protect CO subunits when coadministered with PC may improve its cardioprotection against I/R injury.     

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.     

Opening of mitochondrial K(ATP) channel occurs downstream of PKC-epsilon activation in the mechanism of preconditioning

We examined whether the mitochondrial ATP-sensitive K channel (K(ATP)) is an effector downstream of protein kinase C-epsilon (PKC-epsilon) in the mechanism of preconditioning (PC) in isolated rabbit hearts. PC with two cycles of 5-min ischemia/5-min reperfusion before 30-min global ischemia reduced infarction from 50.3 +/- 6.8% of the left ventricle to 20.3 +/- 3.7%. PC significantly increased PKC-epsilon protein in the particulate fraction from 51 +/- 4% of the total to 60 +/- 4%, whereas no translocation was observed for PKC-delta and PKC-alpha. In mitochondria separated from the other particulate fractions, PC increased the PKC-epsilon level by 50%. Infusion of 5-hydroxydecanoate (5-HD), a mitochondrial K(ATP) blocker, after PC abolished the cardioprotection of PC, whereas PKC-epsilon translocation by PC was not interfered with 5-HD. Diazoxide, a mitochondrial K(ATP) opener, infused 10 min before ischemia limited infarct size to 5.2 +/- 1.4%, but this agent neither translocated PKC-epsilon by itself nor accelerated PKC-epsilon translocation after ischemia. Together with the results of earlier studies showing mitochondrial K(ATP) opening by PKC, the present results suggest that mitochondrial K(ATP)-mediated cardioprotection occurs subsequent to PKC-epsilon activation by PC.     

Orientation of cytochrome c oxidase molecules in the two populations of reconstituted vesicles resolved by column chromatography on DEAE-Sephacryl

Vesicles reconstituted with bovine heart cytochrome c oxidase and dioleoylphosphatidylcholine can be resolved into two populations by column chromatography in DEAE-Sephacryl (Madden, T.D. and Cullis, P.R. (1984) J. Biol. Chem. 259, 7655-7658). These two fractions (I and II) were treated with two proteases. These are trypsin, which has been found to cleave subunit IV in the M domain of the cytochrome c oxidase molecule, and chymotrypsin, which has been found to cleave subunit III in the C domain. These studies show that fraction I vesicles contain cytochrome c oxidase orientation with the M domain outside, i.e., in the same topology as in submitochondrial particles, while fraction II vesicles contain enzyme molecules with their C domain outside, and thus in the same orientation as in mitochondria.     

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.     

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.     

Type A and type B monoamine oxidase activities in rat brain and liver mitochondria: a comparison of their properties using the non-ionic detergent Triton X-100

1. The effects of the non-ionic detergent Triton X-100 on the heterogeneity of monoamine oxidase activities were studied and compared in synaptic (fractions SM and SM2) and non-synaptic (fraction M) brain mitochondria and liver mitochondria. 2. Triton X-100 inhibited type A and type B monoamine oxidase activities in all four mitochondrial fractions in a concentration-dependent manner. Liver mitochondrial enzymatic activities were much more sensitive to this inhibition than those of brain mitochondria. The activities in the SM fraction of synaptic brain mitochondria were the least susceptible. 3. In all four mitochondrial fractions, type A activities were more sensitive to inhibition than type B activities. 4. These results suggest that the membrane micro-environment around the enzyme molecules in situ may be important in the functional expression of the activity of the enzyme.     

The conformational states of cytochrome oxidase in the mitochondrion.

THE Soret spectrum of "resting" cytochrome oxidase in cytochrome-c depleted mitochondria has been determined. The spectrum obtained is dependent on the rate at which the oxidase is turning over. In the least active preparations, the spectrum is almost pure "oxidized" oxidase. With increasing activity the spectrum is converted to a mixture of "oxidized" and "oxygenated" oxidases. It is concluded that the same conformational differences between the two non-reduced forms that are found in the purified enzyme also occur in these cytochrome-c depleted mitochondria.     

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.     

Magnetic interactions in milk xanthine oxidase

The relaxation behavior of the EPR signals of MoV, FAD semiquinone, and the reduced Fe/S I center was measured in the presence and absence of other paramagnetic centers in milk xanthine oxidase. Specific pairs of prosthetic groups were rendered paramagnetic by poising the native enzyme or its desulfo glycol inhibited derivative at appropriate potentials and pH values. Magnetic interactions were found between the following species: Mo--Fe/S I (100-fold increase in microwave power required to saturate the MoV EPR signal at 103 K when Fe/S I is reduced as opposed to oxidized), FAD--Fe/S I and FAD--Fe/S II (70-fold increase in power required to saturate the FADH.EPR signal at 173 K when either Fe/S center is reduced), and Fe/S I--Fe/S II (2.5-fold increase in power to saturate the reduced Fe/S I EPR signal at 20 K when Fe/S II is reduced). The Mo--Fe/S I interaction was also detected as a reduced Fe/S I induced splitting of the MoV EPR spectrum at 30 K. No splittings of the FADH. or Fe/S center spectra were detected. No magnetic interactions were found between FAD and Mo or between Mo and Fe/S II. These results, together with those of Coffman & Buettner [Coffman, R. E., & Buettner, G. R. (1979) J. Phys. Chem. 83, 2392-2400], were used to estimate the following approximate distances between the electron carrying prosthetic groups of milk xamthine oxidase: Mo--Fe/S I, 11 +/- 3 A; Fe/S I-Fe/S II, 15 +/- 4 A; FAD-Fe/S I, 16 +/- 4 A; FAD-Fe/S II, 16 +/- 4 A. A model for the arrangement of these groups within the xanthine oxidase molecule is suggested.     

Trypanosoma brucei: differential requirement of membrane potential for import of proteins into mitochondria in two developmental stages

Trypanosome alternative oxidase (TAO) and the cytochrome oxidase (COX) are two developmentally regulated terminal oxidases of the mitochondrial electron transport chain in Trypanosoma brucei. Here, we have compared the import of TAO and cytochrome oxidase subunit IV (COIV), two stage-specific nuclear encoded mitochondrial proteins, into the bloodstream and procyclic form mitochondria of T. brucei to understand the import processes in two different developmental stages. Under in vitro conditions TAO and COIV were imported and processed into isolated mitochondria from both the bloodstream and procyclic forms. With mitochondria isolated from the procyclic form, the import of TAO and COIV was dependent on the mitochondrial inner membrane potential (delta psi) and required protein(s) on the outer membrane. Import of these proteins also depended on the presence of both internal and external ATP. However, import of TAO and COIV into isolated mitochondria from the bloodstream form was not inhibited after the mitochondrial delta psi was dissipated by valinomycin, CCCP, or valinomycin and oligomycin in combination. In contrast, import of these proteins into bloodstream mitochondria was abolished after the hydrolysis of ATP by apyrase or removal of the ATP and ATP-generating system, suggesting that import is dependent on the presence of external ATP. Together, these data suggest that nuclear encoded proteins such as TAO and COIV are imported in the mitochondria of the bloodstream and the procyclic forms via different mechanism. Differential import conditions of nuclear encoded mitochondrial proteins of T. brucei possibly help it to adapt to different life forms.     

Identification by flow cytometry of two distinct rhodamine-123-stained mitochondrial populations in rat liver

Isolated rat liver mitochondria were split into three fractions of increasing density when applied to a Percoll gradient. NADH-ubiquinone oxidoreductase, succinate dehydrogenase and cytochrome-c oxidase but not F1-ATPase activities increased with density as well as respiratory rate in state 3 and the respiratory control index. Flow cytometry of mitochondrial density fractions stained with rhodamine-123 revealed the occurrence in each density fraction of two distinct mitochondrial populations with different fluorescence intensity. The high fluorescence population was minor and its proportion decreased with density. The extent of high fluorescence population staining depended on the deenergized state of the mitochondria suggesting that this population represents an immature form of the mitochondria which may develop into a fully functional organelle by the incorporation of structural and/or functional proteins.     

Cytokinin control in subcellular localization of indoleacetic acid oxidase and peroxidase

IAA oxidase and peroxidase were found in all subcellular fractions of tobacco callus cells. The bound and cytoplasmic fractions differed greatly in IAA oxidase/peroxidase ratio and in isoperoxidase composition. The IAA oxidase/peroxidase ratio was particularly high in the plasma membrane-rich fraction. Kinetin had profound effects on IAA oxidase and peroxidase. The appearance of fast migrating isoperoxidases in response to 0·2 μM kinetin was found only in cytoplasmic, plasma membrane and ribosome-rich fractions; a high concentration of kinetin inhibited their formation. High kinetin concentrations also lowered the specific activity of IAA oxidase and peroxidase in all subcellular fractions, but the effect was much greater on peroxidase than on IAA oxidase, thus resulting in a drastic increase in IAA oxidase/peroxidase ratio. Evidently the activities of IAA oxidase and peroxidase were not equivalent and should be considered separately.     

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.     

Protein-protein interactions of cytochrome oxidase in inner mitochondrial membranes. The effect of liposome fusion on protein rotational mobility

Rotational diffusion of cytochrome oxidase in the inner membrane of rat liver mitochondria was measured by detecting the decay of absorption anisotropy after photolysis of the heme a3.CO complex by a vertically polarized laser flash. As in previous experiments with beef heart mitochondria (Kawato, S., Sigel, E., Carafoli, E., and Cherry, R. J. (1980) J. Biol. Chem. 255, 5508-5510), co-existence of rotating cytochrome oxidase (mean rotational relaxation time, phi, of 700 to 1400 microseconds) and immobilized cytochrome oxidase (phi greater than 20 ms) was observed in mitochondria and mitoplasts. The effect of lipid/protein ratio by weight (L/P) on the relative proportions of mobile and immobile cytochrome oxidase was investigated following the fusion of soybean phospholipid vesicles with mitoplasts. The fusion procedure yielded four separate fractions upon sucrose density gradient centrifugation with L/P as follows: 0.3 in Pellet, 0.7 in Band 3, 1.5 in Band 2, and 3.0 in Band 1. The percentage of rotationally mobile cytochrome oxidase (phi = 700 to 1000 microseconds) in each of the different bands was found to be 16% in Pellet, 25% in Band 3, 47% in Band 2, and 76% in Band 1 at 37 degrees C. The dependence of the amount of mobile cytochrome oxidase on L/P indicates that the fraction of aggregated protein progressively decreases with decreasing concentration of proteins in the membrane. Thus, the large immobile fraction of cytochrome oxidase in mitochondrial inner membranes can be explained by nonspecific protein aggregation which is a consequence of the low L/P. The decrease in the mobile fraction in Pellet compared with mitoplasts was shown to be due to the pH 6.5 incubation used for fusion.     

Inactivation of cytochrome-c with glucose oxidase

A novel reaction of cytochrome-c from the horse heart with the enzyme glucose oxidase from Aspergillus niger (EC 1.1.3.4), in acidic media is described. Glucose oxidase is able to induce a rapid, profound and irreversible physico-chemical change in cytochrome-c, under anaerobic conditions and in the presence of glucose. The initial rate of reaction is almost independent of the concentration of enzyme and glucose. The striking feature of this reaction is the fact that the reaction proceeds efficiently even below a concentration of 10 nM enzyme.     

The mitochondrial localization of coproporphyrinogen III oxidase.

The location of coproporphyrinogen III oxidase in mitochondria was studied in rat liver by using the digitonin method or hypo-osmotic media for fractionation. The enzyme was found in the intermembrane space with a fraction loosely bound to the inner membrane. This fraction was released by washing the inner-membrane-matrix complex with alkaline solutions or solutions of high ionic strength. The enzyme in both fractions had the same Km (0.16 micrometer) for coproporphyrinogen III. When incubation was performed in a medium that avoided destruction of enzyme membrane binding, a dramatic increase in activity was observed after sonication of whole mitochondria or of the inner-membrane-matrix complex.     

Studies on mouse liver urate oxidase

Summary Distribution of urate oxidase in subcellular components such as nuclei, mitochondria, lysosomes, microsomes, and cell sap, was investigated by both enzymatic and immunochemical methods. The subcellular components were prepared from mouse liver homogenate by differential centrifugation and the resulting microbody-rich mitochondrial fraction was fractionated by sucrose density gradient centrifugation. The enzymatically determined urate oxidase was distributed mainly in mitochondrial and lysosome fractions. The immunochemically assayed urate oxidase antigen was localized in mitochondrial, lysosome, and microsome fractions. The antigen to enzyme ratio was 1.0 in the mitochondrial and lysosome fractions, and about 2.0 in the microsome fraction.Sucrose density gradient centrifugation of the mitochondrial fraction indicated that the urate oxidase antigen was distributed around three density bands of 1.07, 1.15, and 1.24. The main band (1.24) was consistent with the microbody fraction. From these results, it was suggested that a precursor protein (proenzyme) might be located in the microsome fraction.This work was supported in part by a grant 777007 from the Ministry of Education, Japan, in 1972.     

Interaction of milk xanthine oxidase with folic acid. Inhibition of milk xanthine oxidase by folic acid and separation of the enzyme into two fractions on Sepharose 4B/folate gel

Inhibition of xanthine oxidase by folic acid was reexamined after complete removal of the contaminant which was responsible for time-dependent inactivation (Lewis, A. S., Murphy, L., Mcalla, C., Fleary, M., and Purcell, S. (1984) J. Biol. Chem. 259, 12-15; Spector, T., and Ferone, R. (1984) J. Biol. Chem. 259, 10784-10786). From turnover experiments using stopped flow equipment with a limited amount of xanthine and excess oxygen, and from kinetic analyses with an oxygen electrode, folic acid was found to be an inhibitor of xanthine oxidase. The inhibition was competitive with xanthine with a Ki value of 4.2 X 10(-5) M. From the behavior of the enzyme in affinity chromatography using a Sepharose 4B/folate column, folic acid was also confirmed to be a competitive inhibitor of xanthine oxidase. When enzyme which had been pretreated with oxipurinol was applied to the affinity column, two fractions of xanthine oxidase were separated. The first fraction was found to contain the fully active form (double-active dimers) from the analyses of spectral changes on addition of xanthine, oxipurinol titration, and ESR slow signal, whereas the second fraction was assumed to contain mixed dimers and double-inactive dimers. The ratio of the content of the first fraction to that of the second fraction supports the hypothesis that there are three enzyme species and that there is no interaction either in catalytic activity or in sulfuration or desulfuration reactions between the two subunits.