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Cytochrome oxidases are perfect model substrates for analyzing the assembly of multisubunit complexes because the need for cofactor incorporation adds an additional level of complexity to their assembly, [cbb.sub.3]-type cytochrome c oxidases ([cbb.sub.3]-Cox) consist of the catalytic subunit CcoN, the membrane-bound c-type cytochrome subunits CcoO and CcoP, and the CcoQ subunit, which is required for [cbb.sub.3]-Cox stability. Biogenesis of [cbb.sub.3]-Cox proceeds via CcoQP and CcoNO subcomplexes, which assemble into the active [cbb.sub.3]-Cox. Most bacteria expressing [cbb.sub.3]-Cox also contain the ccoGHIS genes, which encode putative [cbb.sub.3]-Cox assembly factors. Their exact function, however, has remained unknown. Here we analyzed the role of CcoH in [cbb.sub.3]-Cox assembly and showed that CcoH is a single spanning-membrane protein with an N-terminus-out--C-terminus-in ([N.sub.out]-[C.sub.in]) topology. In its absence, neither the fully assembled [cbb.sub.3]-Cox nor the CcoQP or CcoNO subcomplex was detectable. By chemical cross-linking, we demonstrated that CcoH binds primarily via its transmembrane domain to the CcoP subunit of [cbb.sub.3]-Cox. A second hydrophobic stretch, which is located at the C terminus of CcoH, appears not to be required for contacting CcoP, but deleting it prevents the formation of the active [cbb.sub.3]-Cox. This suggests that the second hydrophobic domain is required for merging the CcoNO and CcoPQ subcomplexes into the active [cbb.sub.3]-Cox. Surprisingly, CcoH does not seem to interact only transiently with the [cbb.sub.3]-Cox but appears to stay tightly associated with the active, fully assembled complex. Thus, CcoH behaves more like a bona fide subunit of the [cbb.sub.3]-Cox than an assembly factor per se. doi: 10.1128/JB.00988-10

Although Neisseria gonorrhoeae is a prolific source of eight c-type cytochromes, little is known about how its electron transfer pathways to oxygen are organized. In this study, the roles in the respiratory chain to oxygen of cytochromes [c.sub.2], [c.sub.4], and [c.sub.5], encoded by the genes cccA, cycA, and cycB, respectively, have been investigated. Single mutations in genes for either cytochrome [c.sub.4] or [c.sub.5] resulted in an increased sensitivity to growth inhibition by excess oxygen and small decreases in the respiratory capacity of the parent, which were complemented by the chromosomal integration of an ectopic, isopropyl-[beta]-D-thiogalactopyranoside (IPTG)-inducible copy of the cycA or cycB gene. In contrast, a cccA mutant reduced oxygen slightly more rapidly than the parent, suggesting that cccA is expressed but cytochrome [c.sub.2] is not involved in electron transfer to cytochrome oxidase. The deletion of cccA increased the sensitivity of the cycB mutant to excess oxygen but decreased the sensitivity of the cycA mutant. Despite many attempts, a double mutant defective in both cytochromes [c.sub.4] and [c.sub.5] could not be isolated. However, a strain with the ectopically encoded, IPTG-inducible cycB gene with deletions in both cycA and cycB was constructed: the growth and survival of this strain were dependent upon the addition of IPTG, so gonococcal survival is dependent upon the synthesis of either cytochrome [c.sub.4] or [c.sub.5]. These results define the gonococcal electron transfer chain to oxygen in which cytochromes [c.sub.4] and [c.sub.5], but not cytochrome [c.sub.2], provide alternative pathways for electron transfer from the cytochrome [bc.sub.1] complex to the terminal oxidase cytochrome [cbb.sub.3]. doi: 10.1128/JB.00002-10

We examined some of the potential mechanisms lungfish (Protopterus dolloi) use to regulate cytochrome c oxidase (CCO), during metabolic depression. CCO activity was reduced by 67% in isolated liver mitochondria of estivating fish. This was likely accomplished, in part, by the 46% reduction in CCO subunit I protein expression in the liver. No change in the mRNA expression levels of CCO subunits I, II, III, and IV were found in the liver, suggesting CCO is under translational regulation; however, in the kidney, messenger limitation may be a factor as the expression of subunits I and II were depressed (~10-fold) during estivation, suggesting tissue-specific mechanisms of regulation. CCO is influenced by mitochondrial membrane phospholipids, particularly cardiolipin (CL). In P. dolloi, the phospholipid composition of the liver mitochondrial membrane changed during estivation, with a ~2.3-fold reduction in the amount of CL. Significant positive correlations were found between CCO activity and the amount of CL and phosphatidylethanolamine within the mitochondrial membrane. It appears CCO activity is regulated through multiple mechanisms in P. dolloi, and individual subunits of CCO are regulated independently, and in a tissue-specific manner. It is proposed that altering the amount of CL within the mitochondrial membrane may be a means of regulating CCO activity during metabolical depression in the African lungfish, P. dolloi. cardiolipin; mRNA expression; protopterus; metabolic depression; phosphatidylethanolamine doi:10.1152/ajpregu.90815.2008

Cytochrome c oxidase (COX) is composed of 13 subunits, of which COX I, II, and III are encoded by a mitochondrial gene. COX I and II function as the main catalytic components, but the function of COX III is unclear. Because myocardial ischemia affects mitochondrial oxidative metabolism, we hypothesized that COX activity and expression would be affected during postischemic cardiomyopathy. This hypothesis was tested in a monkey model following myocardial infarction (MI) and subsequent pacing-induced heart failure (HF). In this model, COX I protein expression was decreased threefold after MI and fourfold after HF (P < 0.05 vs. sham), whereas COX II expression remained unchanged. COX III protein expression increased 5-fold after MI and further increased 10-fold after HF compared with sham (P < 0.05 vs. sham). The physiological impact of COX III regulation was examined in vitro. Overexpression of COX III in mitochondria of HL-I cells resulted in an 80% decrease in COX I, 60% decrease in global COX activity, 60% decrease in cell viability, and threefold increase in apoptosis (P < 0.05). Oxidative stress induced by [H.sub.2][O.sub.2] significantly (P < 0.05) increased COX III expression. [H.sub.2][O.sub.2] decreased cell viability by 47 [+ or -] 3% upon overexpression of COX III, but only by 12 [+ or -] 5% in control conditions (P < 0.05). We conclude that ischemic stress in vivo and oxidative stress in vitro lead to upregulation of COX III, followed by downregulation of COX I expression, impaired COX oxidative activity, and increased apoptosis. Therefore, upregulation of COX III may contribute to the increased susceptibility to apoptosis following MI and subsequent HF. cytochrome oxidase; mitochondria; myocardial ischemia; oxidative stress doi: 10.1152/ajpcell.00045.2009

Scol and Sco2 are mitochondrial copper-binding proteins involved in the biogenesis of the CUA site in the cytochrome c oxidase (CcO) subunit Cox2 and in the maintenance of cellular copper homeostasis. Human Surfl is a CcO assembly factor with an important but poorly characterized role in CcO biogenesis. Here, we analyzed the impact on CcO assembly and tissue copper levels of a G132S mutation in the juxtamembrane region of SCO1 metallochaperone associated with early onset hypertrophic cardiomyopathy, encephalopathy, hypotonia, and hepatopathy, assessed the total copper content of various SURF1 and SC2-deficient tissues, and investigated the possible physical association between CcO and Scol. The steady-state level of mutant Sco1 was severely decreased in the muscle mitochondria of the SCO1 patient, indicating compromised stability and thus loss of function of the protein. Unlike the wild-type variant, residual mutant Sco1 appeared to migrate exclusively in the monomeric form on blue native gels. Both the activity and content of CcO were reduced in the patient's muscle to ~10-20% of control values. SCO1-deficient mitochondria showed accumulation of two Cox2 subcomplexes, suggesting that Sco1 is very likely responsible for a different posttranslational aspect of Cox2 maturation than Sco2. Intriguingly, the various SURF1-deficient samples analyzed showed a tissue-specific copper deficiency similar to that of SCO-deficient samples, suggesting a role for Surfl in copper homeostasis regulation. Finally, both blue native immunoblot analysis and coimmunoprecipitation revealed that a fraction of Scol physically associates with the CcO complex in human muscle mitochondria, suggesting a possible direct relationship between CcO and the regulation of cellular copper homeostasis. mitochondria; copper; SCO2; SURF1

The trxA gene is regarded as essential in Bacillus subtilis, but the roles of the TrxA protein in this gram-positive bacterium are largely unknown. Inactivation of trxA results in deoxyribonucleoside and cysteine or methionine auxotrophy. This phenotype is expected if the TrxA protein is important for the activity of the class Ib ribonucleotide reductase and adenosine-5'-phosphosulfate/3'-phosphoadenosine-5'-phosphosulfate reductase. We demonstrate here that a TrxA deficiency in addition causes defects in endospore and cytochrome c synthesis. These effects were suppressed by BdbD deficiency, indicating that TrxA in the cytoplasm is the primary electron donor to several different thiol-disulfide oxidoreductases active on the outer side of the B. subtilis cytoplasmic membrane.

The function of the nonreceptor tyrosine kinase c-Src as a plasma membrane-associated molecular effector of a variety of extracellular stimuli is well known. Here, we show that c-Src is also present within mitochondria, where it phosphorylates cytochrome c oxidase (Cox). Deleting the c-src gene reduces Cox activity, and this inhibitory effect is restored by expressing exogenous c-Src. Furthermore, reducing endogenous Src kinase activity down-regulates Cox activity, whereas activating Src has the opposite effect. Src-induced Cox activity is required for normal function of cells that require high levels of ATP, such as mitochondria-rich osteoclasts. The peptide hormone calcitonin, which inhibits osteoclast function, also down-regulates Cox activity. Increasing Src kinase activity prevented the inhibitory effect of calcitonin on Cox activity and osteoclast function. These results suggest that c-Src plays a previously unrecognized role in maintaining cellular energy stores by activating Cox in mitochondria.

Research has been conducted on antimycin-treated chromophores from Rodobacter sphaeroides. Results demonstrate that addition of myxothiazol to these chromophores at ambient redox potentials increases flash-induced oxidation of cytochrome c (sub)1.

Research has been conducted on Rhodobacter sphaeroides heme-copper oxidases with the D-channel. The characterization of the oxidase's mutant has been carried out via the examination of the individual step rates in the catalytic cycle and the results indicate that the nature of the oxygenated intermediates has not been disturbed by the mutant.

Research has been conducted on cytochrome c oxidase. The role of bound Cl (super)- in cytochrome c oxidase's redox-linked protonation kinetics and stoichiometry has been investigated via the use of multiwavelength stopped-flow spectroscopy and the results demonstrate that H (super)+ promotes this protonation and Cl (super)- dissociation.

The kinetics of the cytochrome oxidase possessing full-size C(sub)552, two truncated forms, and a set of site-specific mutants at the docking site of the cytochrome are similar to the wild-type and mitochondrial cytochrome c, and show biphasic kinetic behavior.

Research has been conducted on the electron-transfer from heme a to heme a (sub)3. The rate constant of this transfer and the flash-induced absorbance changes in the fully reduced and mixed-valance enzymes have been investigated and are reported.

We used spontaneously hypertensive rats to study remodeling of cardiac bioenergetics associated with changes in blood pressure. Blood pressure was manipulated with aggressive antihypertensive treatment combining low dietary salt and the angiotensin-converting enzyme inhibitor enalapril. Successive cycles of 2 wk on, 2 wk off treatment led to rapid, reversible changes in left ventricular (LV) mass (30% change in hypertension; mitochondria; mitochondrial deoxyribonucleic acid; angiotensin-converting enzyme inhibitor; spontaneously hypertensive rats; cytochrome oxidase; oxidative stress

Research has been conducted on Paracoccus denitrificans cytochrome c oxidase. The tyrosine vibrational modes in this oxidase have been investigated via the use of spectroscopic FTIR and electrichemical approach and the results indicate that small contributions arise from the tyrosines.

Research has been conducted on the cytochrome c oxidase from Rhodobacter sphaeroides which contains Ca (super)2+ ions. The structural role of Ca (super)2+ -binding site in the bacterial oxidases has been investigated and the results are reported.

Research has been conducted on the heme oxygenase. The crystallization of rat heme oxygenase has been examined and the results of its three-dimensional structure are reported.

Research has been conducted on yeast iso-1-cytochrome c. The reaction of this cytochrome with the low- and high-affinity binding sites on cytochrome c peroxidase compound I has been investigated via the stopped-flow spectroscopy and the details are presented.

Research has been conducted on the heme-copper respiratory oxidases. The redox-linked proton-pumping mechanism of these oxidases have been investigated via the use of cryogenic difference FTIR and time-resolved step-scan Fourier transform infrared spectroscopy and the results are discussed.

The halophilic archaeon Haloferax volcanii possesses a cytochrome whose characteristics resemble a-type cytochromes and contains heme a and a(sub)3. It reduces cytochrome c indicating that it mediates electron transfer reactions.

Endogenously produced nitric oxide (NO) controls oxygen consumption by inhibiting cytochrome c oxidase, the terminal electron acceptor of the mitochondrial electron transport chain. The oxygen-binding site of the enzyme is an iron/copper (haem [a.sub.3]/[Cu.sub.B]) binuclear centre. At high substrate (ferrocytochrome c) concentrations, NO binds reversibly to the reduced iron in competition with oxygen. At low substrate concentrations, NO binds to the oxidized copper. Inhibition at the haem iron site is relieved by dissociation of the NO from the reduced iron. Inhibition at the copper site is relieved by oxidation of the bound NO and subsequent dissociation of nitrite from the enzyme. Therefore, NO can be a substrate, inhibitor or effector of cytochrome oxidase, depending on cellular conditions.

Research has been conducted on the bovine heart cytochrome c oxidase. The proton-transfer reactions on the surface of this oxidase have been investigated via the laser-induced proton-pulse technique and molecular modeling and the results are reported.

Research describes cloning of the cydA and cydB genes that code for the two subunits of the cytochorme bd oxidase in Mycobacterium smegmatis along with the analysis of their expression and phenotypic characterization of the mutants. Data indicate that cytochrome bd oxidase has a role in the respiration of the bacterium.

Research has been conducted on the cytochrome c oxidase. The synthesis of the imidazole-based tridentates which contain -OH or -NHCH (sub)3 intermediates for the cytochrome c oxidase model studies is described.

Research has been conducted on the cytochrome c oxidase from Rhodobacter spaeroides. The reaction of this oxidase with hydrogen peroxide has been investigated under alkaline and acidic conditions via the stopped-flow apparatus.

Research has been conducted on the cytochrome c oxidase. The rapid freeze-quench and electron spin echo envelope modulation analysis of Mn (super)2+ -substitutes cytochrome c oxidase has been carried out to examine the deuterium exchange rate.

Results reveal that the fast electrogenic phase, generated by the electron transfer to heme a, is seen after a single flash on the oxidized cytochrome oxidase. Data indicate that heme a reduction does not accompany proton uptake suggesting that proton uptake is solely a function of heme a reduction.

Fourier transform infrared spectroscopic photolysis difference spectra of the carbon monooxide and cyanide compounds of reduced bovine cytochrome c oxidase reveal that the former is bound in a single form and the latter in at least two forms. Data show that photolysis produces spectral changes in the 1000-1800 wavelength region.

Results show that copperA-domain of cytochrome oxidase subunit II from Paracoccus denitrificans is Ph responsive exhibiting conformational changes at the copperA site with pH decrease. Data further indicate that the protein's conformational state is more stable at the 'low-pH' and the conformational transition is influenced by a slow deprotonation step.

Results show that external ligands can access the heme a(sub)3 of cytochrome c oxidase after its transient coordination to copper(sub)B and likely with both chloride and cyanide bound to the enzyme. Data indicate that in this state, the rate of chloride dissociation exceeds that of spontaneous dissociation.

Results show that the mutant form of cytochrome c oxidase, in which arginine 54 is substituted to methionine, exhibits an impaired electron transfer from Cu(sub)A to heme a. Data further indicate that the reduced enzyme is capable of proton translocation.

The transition of cytochrome c oxidase 'peroxy' intermediate to the ferryl form is regulated by an uptake of one proton without the requirement of any external donor. The group mediating the proton uptake is located in the vicinity of the binuclear center of the 'peroxy' intermediate.

Results show that the C-terminus of the cytochrome c oxidase subunit II from Rhodobacter sphaeroides exhibits cytochrome c binding as revealed by the truncated and histidine tagged protein. The modified protein, upon purification and reconstitution into lipid vesicles, shows an higher proton pumping capacity.

Results reveal that glutamate(I-286) does not function as a 'proton gate' of cytochrome oxidase c proton pumping machinery in mitochondrial-type enzyme systems. Data suggest that the enzymatic machinery does not involve a protonatable/polar residue at any specified site as shown by relocating the glutamate(I286).

Research elucidates the mechanism of proton pump of the cytochrome c oxidase with the demonstration of a cooperative proton-electron coupling at heme a/Cu(sub)A complex. Proton release occurs in the external aqueous phase during oxidation of heme a and copper, whereas during oxidase's rereduction, protons are derived from the inner space.

Results point out that transformation of nitric oxide into nitrite is facilitated by low electron flux through cytochrome c oxidase. At high electron flux, a complex of fully reduced nitric oxide-oxidase is formed.

The extent to which bovine cytochrome c oxidase (COX) dimerizes in nondenaturing detergent environments has been found using sedimentation velocity and equilibrium. It was found that the intrinsic dimeric structure of COX can be maintained only after solubilization with low concentrations of dodecylmaltoside at near neutral pH. Precautions must nevertheless be taken to prevent dissociation into monomers. The structure of the solubilizing detergent is an important factor in detergent effectiveness. It either monomerizes or aggregates.

Structural subunits necessary for formation of the metal centers in subunit I of the cytochrome c oxidase of Rhodobacter sphaeroides have been identified. Using genetic manipulation of the aa(sub.3)-type cytochrome c oxidase of the organism, the minimal structural subunit associations needed for the assembly of the heme A and copper centers of subunit I were determined. Association of subunit II with subunit I is needed for stable formation of the active site in subunit I. Subunit I(sub.a) likely is present in any preparation of the aa(sub.3)-type oxidase isolated via an affinity tag on subunit I.

Self-assembly of heme A and heme B in a designed four-helix bundle have been studied. The single midpoint redox potential of heme B with the split midpoint redox potential of heme A showed the existence of syn and anti topologies of the two di-alpha-helical monomers in the four-helix bundle.

Directed molecular evolution of cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae has been used to generate mutants with increased activity against the classical peroxidase substrate guaiacol. This changed the substrate specificity of CCP from the protein cytochrome c to a small organic molecule. Results indicate that the distal arginine has an important job gatekeeping in the active site of CCP. It controls access to the ferryl oxygen and the distal histidine. Ability of directed molecular evolution technologies to give solutions to biochemical problems that would not be readily predicted by rational design was shown.

Coupling of electron transfer with proton transfer at heme a and Cu(sub.A) (redox Bohr effects ) has been studied in cytochrome c oxidase using carbon monoxide-inhibited enzyme. Results are in agreement with primary involvement of the redox Bohr effects shared by heme a and Cu(sub.A) in the proton-pumping activity of cytochrome c oxidase.

Repeated invasions of fresh water by the copepod Eurytemora affinis have taken place over a short time span, and a study using mitochondrial DNA sequences has been undertaken to reconstruct the geographic and evolutionary history of the invasions. Genetic evidence and physiological support for rapid transitions from saline to fresh water with repeated global-scale invasions has been found.

The mechanisms by which cold exposure alters the expression of cytochrome-c oxidase (CCO) in the cold stenothermal Antarctic eelpout Pachycara brachycephalum and the eurythermal eelpout Zoarces viviparus were investigated. Results suggested that seasonal cold acclimation of Z. viviparus leads to an alteration in the relationship between transcription and translation or posttranslational processes. In the cold-adapted P. brachycephalum, the CCO expression was found to have similar characteristics with that of warm-acclimated confamilial species.

Mutations of SURF-1 in Leigh disease (subacute necrotizing encephalomyelopathy) linked to cytochrome c oxidase (COX) deficiency are discussed. Eleven patients in nine families were studied with mutation analysis of candidate genes which included SURF-1, the yeast homologue of which codes for a mitochondrial protein required for maintenance of COX respiration and activity.

A high rate of parental consanguinity exists in cytochrome-oxidase deficiency, standing at 10%-30%. Studying 157 patients from 148 families it was found that the most common causes of respiratory chain defects in childhood are complex I, complex !V and complex I+IV, at 33%, 28% and 28%, respectively. Autosomal recessive inheritance is involved in most cases of mitochondrial childhood disorders. Genetic counseling would be in order. In North African families a rate of inbreeding higher still was seen. With parental consanguinity related to geographic origin there is a greater effect.

Electron transfer by cytochrome c in the covalent Bacillus (B.) subtilis cytochrome caa3 complex closely resembles the same present in the ionically stabilized mitochondrial complex. The fully reduced cytochrome c oxidase complexes first with O2. This is followed by electron transfer from both cytochrome a and a3. Electron input from Cu(sub A) center and the cytochrome c domain follows quickly. Unlike the mitochondrial complex, reactivity of cytochrome c in caa3 does not vary with ionic strength.

The apparent affinities for oxygen uptake by respiring cells and membranes from mutant without the alternative oxidase cytochrome bo' in Escherichia coli were determined by deoxygenation of oxyleghaemoglobin as a sensitive reporter of dissolved oxygen concentration. Findings indicate that both high-spin haems in the cytochrome bd complex bind CO and show that oxygen can also react with both haems.