This review focuses on the type A cytochrome c oxidases (CcO), which are found in all mitochondria and also in several aerobic bacteria. CcO catalyzes the respiratory reduction of dioxygen (O2) to water by an intriguing mechanism, the details of which are fairly well understood today as a result of research for over four decades. Perhaps even more intriguingly, the membrane-bound CcO couples the O2 reduction chemistry to translocation of protons across the membrane, thus contributing to generation of the electrochemical proton gradient that is used to drive the synthesis of ATP as catalyzed by the rotary ATP synthase in the same membrane. After reviewing the structure of the core subunits of CcO, the active site, and the transfer paths of electrons, protons, oxygen, and water, we describe the states of the catalytic cycle and point out the few remaining uncertainties. Finally, we discuss the mechanism of proton translocation and the controversies in that area that still prevail.

The diarylquinoline TMC207 kills Mycobacterium tuberculosis by specifically inhibiting ATP synthase. We show here that human mitochondrial ATP synthase (50% inhibitory concentration [IC 50 ] of >200 M) displayed more than 20,000-fold lower sensitivity for TMC207 compared to that of mycobacterial ATP synthase (IC 50 of 10 nM). Also, oxygen consumption in mouse liver and bovine heart mitochondria showed very low sensitivity for TMC207. These results suggest that TMC207 may not elicit ATP synthesis-related toxicity in mammalian cells. ATP synthase, although highly conserved between prokaryotes and eukaryotes, may still qualify as an attractive antibiotic target.A new series of compounds, the diarylquinolines, was reported to be highly active against Mycobacterium tuberculosis (3). TMC207, the lead compound of the diarylquinoline series, displays MICs of 30 nM for M. tuberculosis and 15 nM for Mycobacterium smegmatis. We recently demonstrated that TMC207 targets ATP synthase, the enzyme responsible for ATP production by oxidative phosphorylation (11). The inhibition of ATP synthase by TMC207 was highly specific, with a 50% inhibitory concentration (IC 50 ) for this enzyme corresponding to the MIC for bacterial growth inhibition (11). This compound, which efficiently kills replicating as well as dormant mycobacteria (12,18), is currently in clinical development in phase IIb trials in patients with multidrug-resistant tuberculosis.An important factor to consider for a new antibacterial drug is the lack of a eukaryotic homologue of the target, as inhibition of a homologous enzyme could lead to toxicity and safety concerns in humans. In the case of TMC207, the target enzyme ATP synthase is essential for survival in higher organisms, as it supplies cells with the bulk of their ATP via oxidative phosphorylation (20). ATP synthase is evolutionarily strongly conserved among prokaryotes and eukaryotes. Universally, ATP synthesis is coupled to the flow of protons from the intercristae region in mitochondria and the periplasmic space in bacteria to the mitochondrial matrix and the bacterial cytoplasm, respectively. Subunit c of ATP synthase, forming a membrane-spanning oligomer, is essential for this proton transport (8).TMC207 binds to subunit c of mycobacterial ATP synthase (11). Several natural compounds, such as oligomycin and venturicidin, are known to block ATP synthase action by interaction with subunit c. However, these compounds are not selective and inhibit ATP synthase not only in bacteria but also in mitochondria (14,15). This lack of selectivity prevents their clinical usage due to toxicity issues and fatality concerns. Mitochondrial toxicity is a major concern in the clinical development of new drugs, as it may lead to disease conditions, such as pancreatitis, peripheral neuropathy, and cardial or skeletal myopathies (1, 21).Hence, it is of key importance to investigate the selectivity of TMC207 towards mycobacterial ATP synthase compared with that towards mitochondrial ATP synthase.Mycobacterium smegmatis mc 2 ...

Reactive oxygen species (ROS) can trigger a transient burst of mitochondrial ROS production via ROS activation of the mitochondrial permeability transition pore (MPTP), a phenomenon termed ROS-induced ROS release (RIRR). The goal of this study was to investigate if the generation of ROS in a discrete region of a cardiomyocyte could serve to propagate RIRR-mediated mitochondrial depolarizations throughout a cell. Our experiments revealed that localized RIRR activated either RIRR-mediated fluctuations in mitochondrial membrane potential (time period: 3-10 min) or a traveling wave of depolarization of the cell's mitochondria (velocity: approximately 5 microm/min). Both phenomena appeared to be mediated by the mitochondrial permeability transition pore and eventually encompassed the majority of the mitochondrial population of both isolated rat and rabbit cardiomyocytes. Furthermore, depolarization was often reversible; the waves of depolarization were then followed by a rapid (approximately 40 microm/min) repolarization wave of the mitochondria. We show that the RIRR can function to communicate the mitochondrial permeability transition from one mitochondrion to another in the isolated adult cardiomyocyte.

Summary In Paracoccusdenitrificans the aa3‐type cytochrome c oxidase and the bb3‐type quinol oxidase have previously been characterized in detail, both biochemically and genetically. Here we report on the isolation of a genomic locus that harbours the gene cluster ccoNOQP, and demonstrate that it encodes an alternative cbb3‐type cytochrome c oxidase. This oxidase has previously been shown to be specifically induced at low oxygen tensions, suggesting that its expression is controlled by an oxygen‐sensing mechanism. This view is corroborated by the observation that the ccoNOQP gene cluster is preceded by a gene that encodes an FNR homologue and that its promoter region contains an FNR‐binding motif. Biochemical and physiological analyses of a set of oxidase mutants revealed that, at least under the conditions tested, cytochromes aa3, bb3. and cbb3 make up the complete set of terminal oxidases in P. denitrificans. Proton‐translocation measurements of these oxidase mutants indicate that all three oxidase types have the capacity to pump protons. Previously, however, we have reported decreased H+/e coupling efficiencies of the cbb3‐type

Fatty-acid metabolism plays a key role in acquired and inborn metabolic diseases. To obtain insight into the network dynamics of fatty-acid β-oxidation, we constructed a detailed computational model of the pathway and subjected it to a fat overload condition. The model contains reversible and saturable enzyme-kinetic equations and experimentally determined parameters for rat-liver enzymes. It was validated by adding palmitoyl CoA or palmitoyl carnitine to isolated rat-liver mitochondria: without refitting of measured parameters, the model correctly predicted the β-oxidation flux as well as the time profiles of most acyl-carnitine concentrations. Subsequently, we simulated the condition of obesity by increasing the palmitoyl-CoA concentration. At a high concentration of palmitoyl CoA the β-oxidation became overloaded: the flux dropped and metabolites accumulated. This behavior originated from the competition between acyl CoAs of different chain lengths for a set of acyl-CoA dehydrogenases with overlapping substrate specificity. This effectively induced competitive feedforward inhibition and thereby led to accumulation of CoA-ester intermediates and depletion of free CoA (CoASH). The mitochondrial [NAD+]/[NADH] ratio modulated the sensitivity to substrate overload, revealing a tight interplay between regulation of β-oxidation and mitochondrial respiration.