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Alternative complex III (ACIII) is a key component of the respiratory and/or photosynthetic electron transport chains of many bacteria.sup.1-3. Like complex III (also known as the bc.sub.1 complex), ACIII catalyses the oxidation of membrane-bound quinol and the reduction of cytochrome c or an equivalent electron carrier. However, the two complexes have no structural similarity.sup.4-7. Although ACIII has eluded structural characterization, several of its subunits are known to be homologous to members of the complex iron-sulfur molybdoenzyme (CISM) superfamily.sup.8, including the proton pump polysulfide reductase.sup.9,10. We isolated the ACIII from Flavobacterium johnsoniae with native lipids using styrene maleic acid copolymer.sup.11-14, both as an independent enzyme and as a functional 1:1 supercomplex with an aa.sub.3-type cytochrome c oxidase (cyt aa.sub.3). We determined the structure of ACIII to 3.4 Å resolution by cryo-electron microscopy and constructed an atomic model for its six subunits. The structure, which contains a [3Fe-4S] cluster, a [4Fe-4S] cluster and six haem c units, shows that ACIII uses known elements from other electron transport complexes arranged in a previously unknown manner. Modelling of the cyt aa.sub.3 component of the supercomplex revealed that it is structurally modified to facilitate association with ACIII, illustrating the importance of the supercomplex in this electron transport chain. The structure also resolves two of the subunits of ACIII that are anchored to the lipid bilayer with N-terminal triacylated cysteine residues, an important post-translational modification found in numerous prokaryotic membrane proteins that has not previously been observed structurally in a lipid bilayer.The structure of alternative complex III, a key enzyme in the bacterial electron transport chain, is reported both alone and as a supercomplex with an aa.sub.3-type cytochrome c oxidase., Author(s): Chang Sun [sup.1] [sup.2] , Samir Benlekbir [sup.3] , Padmaja Venkatakrishnan [sup.1] [sup.8] , Yuhang Wang [sup.1] [sup.2] [sup.4] , Sangjin Hong [sup.1] , Jonathan Hosler [sup.5] , Emad [...]

Cytochrome c oxidase is a member of the haem copper oxidase superfamily (HCO) (1). HCOs function as the terminal enzymes in the respiratory chain of mitochondria and aerobic prokaryotes, coupling [...]

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain that catalyzes respiratory reduction of dioxygen ([O.sub.2]) to water in all eukaryotes and many aerobic bacteria. CcO, and its homologs among the heme-copper oxidases, has an active site composed of an oxygen-binding heme and a copper center in the vicinity, plus another heme group that donates electrons to this site. In most oxidoreduction enzymes, electron transfer (eT) takes place by quantum-mechanical electron tunneling. Here we show by independent molecular dynamics and quantum-chemical methods that the heme-heme eT in CcO differs from the majority of cases in having an exceptionally low reorganization energy. We show that the rate of interheme eT in CcO may nevertheless be predicted by the Moser-Dutton equation if reinterpreted as the average of the eT rates between all individual atoms of the donor and acceptor weighed by the respective packing densities between them. We argue that this modification may be necessary at short donor/ acceptor distances comparable to the donor/acceptor radii. biological electron transfer | heme-copper oxidases | Moser-Dutton ruler | reorganization energy doi/ 10.1073/pnas.1005889107

Kinetic studies of heme-copper terminal oxidases using the CO flow-flash method are potentially compromised by the fate of the photodissociated CO. In this time-resolved optical absorption study, we compared the kinetics of dioxygen reduction by [ba.sub.3] cytochrome c oxidase from Thermus thermophilus in the absence and presence of CO using a photolabile [O.sub.2]-carrier. A novel double-laser excitation is introduced in which dioxygen is generated by photolyzing the [O.sub.2]-carrier with a 355 nm laser pulse and the fully reduced CO-bound ba3 simultaneously with a second 532-nm laser pulse. A kinetic analysis reveals a sequential mechanism in which [O.sub.2] binding to heme [a.sub.3] at 90 [micro]M [O.sub.2] occurs with lifetimes of 9.3 and 110 [micro]s in the absence and presence of CO, respectively, followed by a faster cleavage of the dioxygen bond (4.8 [micro]s), which generates the P intermediate with the concomitant oxidation of heme b. The second-order rate constant of 1 x [10.sup.9][M.sup.-1][s.sup.-1] for [O.sub.2] binding to ba3 in the absence of CO is 10 times greater than observed in the presence of CO as well as for the bovine heart enzyme. The [O.sub.2] bond cleavage in [ba.sub.3] of 4.8 [micro]s is also approximately 10 times faster than in the bovine enzyme. These results suggest important structural differences between the accessibility of [O.sub.2] to the active site in ba3 and the bovine enzyme, and they demonstrate that the photodissociated CO impedes access of dioxygen to the heme a3 site in [ba.sub.3], making the CO flow-flash method inapplicable. double-laser technique | T. thermophilus [ba.sub.3] | oxygen reduction | slow-fast kinetics | [O.sub.2] channel doi/ 10.1073/pnas.1008603107

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

The heme-copper oxidases (HCOs) accomplish the key event of aerobic respiration; they couple [O.sub.2] reduction and transmembrane proton pumping. To gain new insights into the still enigmatic process, we structurally characterized a C-family HCO--essential for the pathogenicity of many bacteria--that differs from the two other HCO families, A and B, that have been structurally analyzed. The x-ray structure of the C-family [cbb.sub.3] oxidase from Pseudomonas stutzeri at 3.2 angstrom resolution shows an electron supply system different from families A and B. Like family-B HCOs, C HCOs have only one pathway, which conducts protons via an alternative tyrosine-histidine cross-link. Structural differences around heroes b and b3 suggest a different redox-driven proton-pumping mechanism and provide clues to explain the higher activity of family-C HCOs at tow oxygen concentrations. 10.1126/science.1187303

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

During the course of evolution, a massive reduction of the mitochondrial genome content occurred that was associated with transfer of a large number of genes to the nucleus. To further characterize factors that control the mitochondrial gene transfer/retention process, we have investigated the barriers to transfer of yeast COX2, a mitochondrial gene coding for a subunit of cytochrome c oxidase complex. Nuclear-recoded Saccharomyces cerevisiae COX2 fused at the amino terminus to various alternative mitochondrial targeting sequences (MTS) fails to complement the growth defect of a yeast strain with an inactivated mitochondrial COX2 gene, even though it is expressed in cells. Through random mutagenesis of one such hybrid MTS-COX2, we identified a single mutation in the first Cox2 transmembrane domain (W56 [right arrow] R) that (i) results in the cellular expression of a Cox2 variant with a molecular mass indicative of MTS cleavage, which (ii) supports growth of a cox2 mutant on a nonfermentable carbon source, and that (iii) partially restores cytochrome c oxidase-specific respiration by the mutant mitochondria. [COX2.sup.W56R] can be allotopically expressed with an MTS derived from S. cerevisiae OXA1 or Neurospora crassa SU9, both coding for hydrophobic mitochondrial proteins, but not with an MTS derived from the hydrophilic protein Cox4. In contrast to some other previously transferred genes, allotopic COX2 expression is not enabled or enhanced by a 3'-UTR that localizes mRNA translation to the mitochondria, such as yeast [ATP2.sup.3'-UTR]. Application of in vitro evolution strategies to other mitochondrial genes might ultimately lead to yeast entirely lacking the mitochondrial genome, but still possessing functional respiratory capacity. cytochrome coxidase | mitochondria | Saccharomyces cerevisiae doi/ 10.1073/pnas.1000735107

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

The toxic gas [H.sub.2]S is produced by enzymes in the body. At moderate concentrations, [H.sub.2]S elicits physiological effects similar to hibernation. Herein, we describe experiments that imply that the phenomenon probably results from reversible inhibition of the enzyme cytochrome c oxidase (CcO), which reduces oxygen during respiration. A functional model of the oxygen-reducing site in CcO was used to explore the effects of [H.sub.2]S during respiration. Spectroscopic analyses showed that the model binds two molecules of H2S. The electro-catalytic reduction of oxygen is reversibly inhibited by [H.sub.2]S concentrations similar to those that induce hibernation. This phenomenon derives from a weak, reversible binding of [H.sub.2]S to the [Fe.sup.II] porphyrin, which mimics heme [a.sub.3] in CcO's active site. No inhibition of CcO is detected at lower [H.sub.2]S concentrations. Nevertheless, at lower concentrations, [H.sub.2]S could have other biological effects on CcO. For example, [H.sub.2]S rapidly reduces [Fe.sup.III] and [Cu.sup.II] in both the oxidized form of this functional model and in CcO itself. [H.sub.2]S also reduces CcO's biological reductant, cytochrome c, which normally derives its reducing equivalents from food metabolism. Consequently, it is speculated that [H.sub.2]S might also serve as a source of electrons during periods of hibernation when food supplies are low. cytochrome c oxidase | reversible inhibition | synthetic functional model | H2S complex doi/10.1073/pnas.0904082106

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

Cytochrome c oxidase (CcO), the terminal enzyme of the respiratory chain, reduces oxygen to water and uses the released energy to pump protons across a membrane. Here, we use kinetic master equations to explore the energetic and kinetic control of proton pumping in CcO. We construct models consistent with thermodynamic principles, the structure of CcO, experimentally known proton affinities, and equilibrium constants of intermediate reactions. The resulting models are found to capture key properties of CcO, including the midpoint redox potentials of the metal centers and the electron transfer rates. We find that coarse-grained models with two proton sites and one electron site can pump one proton per electron against membrane potentials exceeding 100 mV. The high pumping efficiency of these models requires strong electrostatic couplings between the proton loading (pump) site and the electron site (heme a), and kinetic gating of the internal proton transfer. Gating is achieved by enhancing the rate of proton transfer from the conserved Glu-242 to the pump site on reduction of heme a, consistent with the predictions of the water-gated model of proton pumping. The model also accounts for the phenotype of D-channel mutations associated with loss of pumping but retained turnover. The fundamental mechanism identified here for the efficient conversion of chemical energy into an electrochemical potential should prove relevant also for other molecular machines and novel fuel-cell designs. bioenergetics | biological machines | kinetic master equation | respiration

Five iron porphyrins with different superstructures were immobilized on self-assembled-monolayer (SAM)-coated interdigitated-array (IDAs) gold-platinum electrodes. The selectivity of the catalysts i.e., limited formation of partially reduced oxygen species (PROS) in the electrocatalytic reduction of dioxygen, is a function of 2 rates: (i) the rate of electron transfer from the electrode to the catalyst, which is controlled by the length, and conjugation of the linker from the catalyst to the electrode and (ii) the rate of bound oxygen (superoxide) hydrolysis, which correlates with the presence of a water cluster in the gas-binding pocket influencing the rate of oxygen binding; these factors are controlled by the nature of the porphyrin superstructure. The structurally biomimetic Trisimidazole model is the most selective. electron transfer rate | oxygen binding rates | water cluster | oxygen complex stabilization | partially reduced oxygen species

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.

Recently, it has been reported that mitochondria possess a novel pathway for nitric oxide (NO) synthesis. This pathway is induced when cells experience hypoxia, is nitrite (N[O.sub.2.sup.-])-dependent, is independent of NO synthases, and is catalyzed by cytochrome c oxidase (Cco). It has been proposed that this mitochondrially produced NO is a component of hypoxic signaling and the induction of nuclear hypoxic genes. In this study, we examine the N[O.sub.2.sup.-]-dependent NO production in yeast engineered to contain alternative isoforms, Va or Vb, of Cco subunit V. Previous studies have shown that these isoforms have differential effects on oxygen reduction by Cco, and that their genes (COX5a and COX5b, respectively) are inversely regulated by oxygen. Here, we find that the Vb isozyme has a higher turnover rate for NO production than the Va isozyme and that the Vb isozyme produces NO at much higher oxygen concentrations than the Va isozyme. We have also found that the hypoxic genes CYC7 and OLE1 are induced to higher levels in a strain carrying the Vb isozyme than in a strain carrying the Va isozyme. Together, these results demonstrate that the subunit V isoforms have differential effects on N[O.sub.2.sup.-]-dependent NO production by Cco and provide further support for a role of Cco in hypoxic signaling. These findings also suggest a positive feedback mechanism in which mitochondrially produced NO induces expression of COX5b, whose protein product then functions to enhance the ability of Cco to produce NO in hypoxic/anoxic cells. hypoxia | mitochondria | reactive oxygen species | nitrite | yeast

One of the many routes proposed for the cellular inactivation of endogenous nitric oxide (NO) is by the cytochrome c oxidase of the mitochondrial respiratory chain. We have studied this possibility in human embryonic kidney cells engineered to generate controlled amounts of NO. We have used visible light spectroscopy to monitor continuously the redox state of cytochrome c oxidase in an oxygen-tight chamber, at the same time as which we measure cell respiration and the concentrations of oxygen and NO. Pharmacological manipulation of cytochrome c oxidase indicates that this enzyme, when it is in turnover and in its oxidized state, inactivates physiological amounts of NO, thus regulating its intra- and extracellular concentrations. This inactivation is prevented by blocking the enzyme with inhibitors, including NO. Furthermore, when cells generating low concentrations of NO respire toward hypoxia, the redox state of cytochrome c oxidase changes from oxidized to reduced, leading to a decrease in NO inactivation. The resultant increase in NO concentration could explain hypoxic vasodilation. hypoxia | mitochondrial respiration | nitric oxide inactivation | nitric oxide synthase

Defects in the mitochondrial cytochrome c oxidase (COX) have been associated with Alzheimer's Disease, in which the age-dependent accumulation of [beta]-amyloid plays an important role in synaptic dysfunction and neurodegeneration. To test the possibility that age-dependent decline in the mitochondrial respiratory function, especially COX activity, may participate in the formation and accumulation of [beta]-amyloid, we generated mice expressing mutant amyloid precursor protein and mutant presenilin 1 in a neuron-specific COX-deficient background. A neuron-specific COX-deficient mouse was generated by the Cre-IoxP system, in which the COX10 gene was deleted by a CamKII[alpha] promoter-driven Cre-recombinase. COX10 is a farnesyltransferase involved in the biosynthesis of heme a, required for COX assembly and function. These KO mice showed an age-dependent COX deficiency in the cerebral cortex and hippocampus. Surprisingly, COX10 KO mice exhibited significantly fewer amyloid plaques in their brains compared with the COX-competent transgenic mice. This reduction in amyloid plaques in the KO mouse was accompanied by a reduction in A[beta]42 level, [beta]-secretase activity, and oxidative damage. Likewise, production of reactive oxygen species from cells with partial COX activity was not elevated. Collectively, our results suggest that, contrary to previous models, a defect in neuronal COX does not increase oxidative damage nor predispose for the formation of amyloidgenic amyloid precursor protein fragments. mitochondria | oxidative phosphorylation | neurodegeneration

Methanol intoxication produces toxic injury to the retina and optic nerve, resulting in blindness. The toxic metabolite in methanol intoxication is formic acid, a mitochondrial toxin known to inhibit the essential mitochondrial enzyme, cytochrome oxidase. Photobiomodulation by red to near-IR radiation has been demonstrated to enhance mitochondrial activity and promote cell survival in vitro by stimulation of cytochrome oxidase activity. The present studies were undertaken to test the hypothesis that exposure to monochromatic red radiation from light-emitting diode (LED) arrays would protect the retina against the toxic actions of methanol-derived formic acid in a rodent model of methanol toxicity. Using the electroretinogram as a sensitive indicator of retinal function, we demonstrated that three brief (2 min, 24 s) 670-nm LED treatments (4 J/[cm.sup.2]), delivered at 5, 25, and 50 h of methanol intoxication, attenuated the retinotoxic effects of methanol-derived formate. Our studies document a significant recovery of rod- and cone-mediated function in LED-treated, methanol-intoxicated rats. We further show that LED treatment protected the retina from the histopathologic changes induced by methanol-derived formate. These findings provide a link between the actions of monochromatic red to near-IR light on mitochondrial oxidative metabolism in vitro and retinoprotection in vivo. They also suggest that photobiomodulation may enhance recovery from retinal injury and other ocular diseases in which mitochondrial dysfunction is postulated to play a role.

Recent work shows that S-adenosylmethionine (AdoMet) helps maintain normal liver function as chronic hepatic deficiency results in spontaneous development of steatohepatitis and hepatocellular carcinoma. The mechanisms by which these nontraditional functions of AdoMet occur are unknown. Here, we use knockout mice deficient in hepatic AdoMet synthesis ([MAT1A.sup.-/-]) to study the proteome of the liver during the development of steatohepatitis. One hundred and seventeen protein spots, differentially expressed during the development of steatohepatitis, were selected and identified by peptide mass fingerprinting. Among them, 12 proteins were found to be affected from birth, when [MAT1A.sup.-/-] expression is switched on in WT mouse liver, to the rise of histological lesions, which occurs at [approximately equal to] 8 months. Of the 12 proteins, 4 [prohibitin 1 (PHB1), cytochrome c oxidase I and II, and ATPase [beta]-subunit] have known roles in mitochondrial function. We show that the alteration in expression of PHB1 correlates with a loss of mitochondrial function. Experiments in isolated rat hepatocytes indicate that AdoMet regulates PHB1 content, thus suggesting ways by which steatohepatitis may be induced. Importantly, we found the expression of these mitochondrial proteins was abnormal in ob/ob mice and obese patients who are at risk for nonalcoholic steatohepatitis.

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.

Geographically isolated populations may accumulate alleles that function well on their own genetic backgrounds but poorly on the genetic backgrounds of other populations. Consequently, interpopulation hybridization may produce offspring of low fitness as a result of incompatibilities arising in allopatry. Genes participating in these epistatic incompatibility systems remain largely unknown. In fact, despite the widely recognized importance of epistatic interactions among gene products, few data directly address the functional consequences of such interactions among natural genetic variants. In the marine copepod, Tigriopus californicus, we found that the cytochrome c variants isolated from two different populations each had significantly higher activity with the cytochrome c oxidase derived from their respective source population. Three amino acid substitutions in the cytochrome c protein appear to be sufficient to confer population specificity. These results suggest that electron transport system (ETS) proteins form coadapted sets of alleles within populations and that disruption of the coadapted ETS gene complex leads to functional incompatibilities that may lower hybrid fitness.

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.

This article discusses the relationship between chronic low frequency stimulation, muscle composition and aging. An analysis of muscle mass, muscle fiber, and cytochrome oxidase is presented.

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 unsymmetrical picket-fence naphthylporphyrin ligands and their metalated porphyrinato complexes. The synthesis of these compounds as precursor models has been carried out for the binuclear cytochrome c oxidase active-site and the results are reported.

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.