SUMMARY T cells undergo metabolic reprogramming with major changes in cellular energy metabolism during activation. In patients with mitochondrial disease, clinical data were marked by frequent infections and immunodeficiency, prompting us to explore the consequences of oxidative phosphorylation dysfunction in T cells. Since cytochrome c oxidase (COX) is a critical regulator of OXPHOS, we created a mouse model with isolated dysfunction in T cells by targeting a gene, COX10, that produces mitochondrial disease in humans. COX dysfunction resulted in increased apoptosis following activation in vitro and immunodeficiency in vivo. Select T cell effector subsets were particularly affected; this could be traced to their bioenergetic requirements. In summary, the findings presented herein emphasize the role of COX particularly in T cells as a metabolic checkpoint for cell fate decisions following T cell activation, with heterogeneous effects in T cell subsets. In addition, our studies highlight the utility of translational models that recapitulate human mitochondrial disease for understanding immunometabolism.

We have optimized a method to directly measure oxygen consumption in acutely isolated, ex vivo mouse retina and demonstrate that photoreceptors have low mitochondrial reserve capacity. Our data provide a plausible explanation for the high vulnerability of photoreceptors to altered energy homeostasis caused by mutations or metabolic challenges.

The concentration of mitochondrial oxidative phosphorylation complexes (MOPCs) is tuned to the maximum energy conversion requirements of a given tissue; however, whether the activity of MOPCs is altered in response to acute changes in energy conversion demand is unclear. We hypothesized that MOPCs activity is modulated by tissue metabolic stress to maintain the energy-metabolism homeostasis. Metabolic stress was defined as the observed energy conversion rate/maximum energy conversion rate. The maximum energy conversion rate was assumed to be proportional to the concentration of MOPCs, as determined with optical spectroscopy, gel electrophoresis, and mass spectrometry. The resting metabolic stress of the heart and liver across the range of resting metabolic rates within an allometric series (mouse, rabbit, and pig) was determined from MPOCs content and literature respiratory values. The metabolic stress of the liver was high and nearly constant across the allometric series due to the proportional increase in MOPCs content with resting metabolic rate. In contrast, the MOPCs content of the heart was essentially constant in the allometric series, resulting in an increasing metabolic stress with decreasing animal size. The MOPCs activity was determined in native gels, with an emphasis on Complex V. Extracted MOPCs enzyme activity was proportional to resting metabolic stress across tissues and species. Complex V activity was also shown to be acutely modulated by changes in metabolic stress in the heart, in vivo and in vitro. The modulation of extracted MOPCs activity suggests that persistent posttranslational modifications (PTMs) alter MOPCs activity both chronically and acutely, specifically in the heart. Protein phosphorylation of Complex V was correlated with activity inhibition under several conditions, suggesting that protein phosphorylation may contribute to activity modulation with energy metabolic stress. These data are consistent with the notion that metabolic stress modulates MOPCs activity in the heart.

The cytochrome bc complexes found in mitochondria, chloroplasts and many bacteria play critical roles in their respective electron transport chains. The quinol oxidase (Q o ) site in this complex oxidizes a hydroquinone (quinol), reducing two one-electron carriers, a low potential cytochrome b heme and the "Rieske" ironsulfur cluster. The overall electron transfer reactions are coupled to transmembrane translocation of protons via a "Q-cycle" mechanism, which generates proton motive force for ATP synthesis. Since semiquinone intermediates of quinol oxidation are generally highly reactive, one of the key questions in this field is: how does the Q o site oxidize quinol without the production of deleterious side reactions including superoxide production? We attempt to test three possible general models to account for this behavior: 1) The Q o site semiquinone (or quinol-imidazolate complex) is unstable and thus occurs at a very low steady-state concentration, limiting O 2 reduction; 2) the Q o site semiquinone is highly stabilized making it unreactive toward oxygen; and 3) the Q o site catalyzes a quantum mechanically coupled two-electron/two-proton transfer without a semiquinone intermediate. Enthalpies of activation were found to be almost identical between the uninhibited Q-cycle and superoxide production in the presence of antimycin A in wild type. This behavior was also preserved in a series of mutants with altered driving forces for quinol oxidation. Overall, the data support models where the rate-limiting step for both Q-cycle and superoxide production is essentially identical, consistent with model 1 but requiring modifications to models 2 and 3.The cytochrome (cyt) 2 bc 1 complex (EC 1.10.2.2) (cyt bc 1 complex) is found on the inner membrane of mitochondria and energy transducing membranes of many bacteria (1-3). It is structurally and functionally homologous to a taxonomically wide spread group, collectively referred to as "bc complexes," additionally consisting of the cyt b 6 f complex and the menaquinol oxidizing complexes in many bacteria (4 -6). In all well studied cases, cyt bc complexes couple the oxidation of a substrate quinol (QH 2 ) to the formation of a proton motive force across the energy transducing membrane in which the given complex resides, the energy of which is ultimately stored as ATP.Cytochrome bc 1 complexes contain four redox-active metal centers, arranged in two separate chains (2, 5, 7-9). The "high potential chain" consists of the Rieske iron-sulfur [2Fe2S] cluster (in the Rieske, or "iron-sulfur subunit," hereafter "the [2Fe2S] cluster"), and a c-type cyt, known in mitochondria and Gram-negative bacteria as cyt c 1 . The "low potential chain," which is liganded to the cyt b subunit of ubiquinol oxidizing complexes, consists of two b-type hemes, cyt b L and b H labeled for their relatively lower and higher electrochemical potentials. Crystal structures from several taxonomic sources (2, 8, 10, 11) suggest a well conserved placement of the cofactors throughout the various cyt bc ...

We have investigated the interaction between monomers of the dimeric yeast cytochrome bc 1 complex by analyzing the pre-steady and steady state activities of the isolated enzyme in the presence of antimycin under conditions that allow the first turnover of ubiquinol oxidation to be observable in cytochrome c 1 reduction. At pH 8.8, where the redox potential of the iron-sulfur protein is ϳ200 mV and in a bc 1 complex with a mutated iron-sulfur protein of equally low redox potential, the amount of cytochrome c 1 reduced by several equivalents of decyl-ubiquinol in the presence of antimycin corresponded to only half of that present in the bc 1 complex. Similar experiments in the presence of several equivalents of cytochrome c also showed only half of the bc 1 complex participating in quinol oxidation. The extent of cytochrome b reduced corresponded to two b H hemes undergoing reduction through one center P per dimer, indicating electron transfer between the two cytochrome b subunits. Antimycin stimulated the ubiquinolcytochrome c reductase activity of the bc 1 complex at low inhibitor/enzyme ratios. This stimulation could only be fitted to a model in which half of the bc 1 dimer is inactive when both center N sites are free, becoming active upon binding of one center N inhibitor molecule per dimer, and there is electron transfer between the cytochrome b subunits of the dimer. These results are consistent with an alternating half-of-the-sites mechanism of ubiquinol oxidation in the bc 1 complex dimer.The cytochrome bc 1 complex transfers electrons from ubiquinol to cytochrome c by the protonmotive Q cycle mechanism (1) in which there are two substrate-binding sites where ubiquinol is oxidized (center P) and ubiquinone is re-reduced (center N). Crystal structures of the bc 1 complexes obtained from various sources (2-4) show a dimeric structure in which the two ironsulfur protein subunits span both monomers in an intertwined arrangement. Crystal structures from the yeast bc 1 complex show ubiquinone at center N of only one monomer and cytochrome c also in only one monomer, suggesting a functional asymmetry in the dimer (5).Some center P inhibitors have been shown to completely block bc 1 complex activity upon binding to only half of the dimeric complex (6), suggesting anti-cooperative interaction between the ubiquinol oxidation sites in the dimer. Antimycin, a center N inhibitor, binds to only one center N of the dimeric enzyme in a mutant where the iron-sulfur cluster cannot be inserted into the Rieske protein, suggesting conformational interaction between centers P and N of different monomers (7). In addition, it has long been observed that titration of the cytochrome c reductase activity of the enzyme with antimycin yields non-linear curves (8, 9), which is unexpected for a tightly bound inhibitor. This anomalous behavior has been attributed to rapid mobility of the inhibitor between the two center N sites in the dimer (10), although an alternate explanation could be equilibration of electrons between the cytochro...

We previously proposed that the dimeric cytochrome bc 1 complex exhibits half-of-the-sites reactivity for ubiquinol oxidation and rapid electron transfer between bc 1 monomers (Covian, R., Kleinschroth, T., Ludwig, B., and Trumpower, B. L. (2007) J. Biol. Chem. 282, 22289 -22297). Here, we demonstrate the previously proposed half-of-the-sites reactivity and intermonomeric electron transfer by characterizing the kinetics of ubiquinol oxidation in the dimeric bc 1 complex from Paracoccus denitrificans that contains an inactivating Y147S mutation in one or both cytochrome b subunits. The enzyme with a Y147S mutation in one cytochrome b subunit was catalytically fully active, whereas the activity of the enzyme with a Y147S mutation in both cytochrome b subunits was only 10 -16% of that of the enzyme with fully wild-type or heterodimeric cytochrome b subunits. Enzyme with one inactive cytochrome b subunit was also indistinguishable from the dimer with two wild-type cytochrome b subunits in rate and extent of reduction of cytochromes b and c 1 by ubiquinol under pre-steady-state conditions in the presence of antimycin. However, the enzyme with only one mutated cytochrome b subunit did not show the stimulation in the steady-state rate that was observed in the wildtype dimeric enzyme at low concentrations of antimycin, confirming that the half-of-the-sites reactivity for ubiquinol oxidation can be regulated in the wild-type dimer by binding of inhibitor to one ubiquinone reduction site.The cytochrome bc 1 complex is a multisubunit enzyme that generates a transmembrane protonmotive force by transferring electrons from ubiquinol to cytochrome c. Energy conservation in this enzyme complex is ensured by the oxidation of ubiquinol and the reduction of ubiquinone at active sites located on opposite sides of the membrane, as described by the protonmotive Q-cycle (1). High resolution structures have shown that the bc 1 complex is a dimer composed of two copies of cytochrome b, the Rieske iron-sulfur protein, and cytochrome c 1 , which are the only polypeptides present in some bacterial enzymes (2), in addition to six to eight additional subunits present exclusively in each monomer of the eukaryotic complex (3-5).The functional relevance of this dimeric arrangement has been supported by an extensive body of kinetic evidence (for review, see Ref. 6), which includes the key observations that only one ubiquinol oxidation site, or center P, is active when the two ubiquinone reduction, or center N, sites are occupied by inhibitors (7), that electrons rapidly equilibrate between the cytochrome b subunits (8, 9), and that there is conformational communication between center P and center N sites (10). We have proposed that this half-of-the sites activity at center P also exists under normal steady-state conditions in the absence of inhibitors and that this mechanism minimizes the leakage of electrons to oxygen under conditions that would favor the accumulation of electrons in the cytochrome b hemes (8, 11). Evidence consistent with this...

The mitochondrial cytochrome bc 1 complex (ubiquinol/cytochrome c oxidoreductase) is generally thought to generate superoxide anion that participates in cell signaling and contributes to cellular damage in aging and degenerative disease. However, the isolated, detergent-solubilized bc 1 complex does not generate measurable amounts of superoxide except when inhibited by antimycin. In addition, indirect measurements of superoxide production by cells and isolated mitochondria have not clearly resolved the contribution of the bc 1 complex to the generation of superoxide by mitochondria in vivo, nor did they establish the effect, if any, of membrane potential on superoxide formation by this enzyme complex. In this study we show that the yeast cytochrome bc 1 complex does generate significant amounts of superoxide when reconstituted into phospholipid vesicles. The rate of superoxide generation by the reconstituted bc 1 complex increased exponentially with increased magnitude of the membrane potential, a finding that is compatible with the suggestion that membrane potential inhibits electron transfer from the cytochrome b L to b H hemes, thereby promoting the formation of a ubisemiquinone radical that interacts with oxygen to generate superoxide. When the membrane potential was further increased, by the addition of nigericin or by the imposition of a diffusion potential, the rate of generation of superoxide was further accelerated and approached the rate obtained with antimycin. These findings suggest that the bc 1 complex may contribute significantly to superoxide generation by mitochondria in vivo, and that the rate of superoxide generation can be controlled by modulation of the mitochondrial membrane potential.The mitochondrial oxidative phosphorylation system utilizes the energy derived from the oxidation of metabolic substrates to drive the synthesis of ATP. Electron transport through the NADH dehydrogenase complex, cytochrome bc 1 complex, and cytochrome c oxidase complex is coupled to proton translocation across the mitochondrial inner membrane, thus generating a protonmotive force (⌬p) consisting of a membrane potential (⌬⌿) and a pH gradient (⌬pH) that drives the synthesis of ATP by the ATP synthase (reviewed in Ref.

Covian R, Balaban RS. Cardiac mitochondrial matrix and respiratory complex protein phosphorylation. Am J Physiol Heart Circ Physiol 303: H940 -H966, 2012. First published August 10, 2012; doi:10.1152/ajpheart.00077.2012.-It has become appreciated over the last several years that protein phosphorylation within the cardiac mitochondrial matrix and respiratory complexes is extensive. Given the importance of oxidative phosphorylation and the balance of energy metabolism in the heart, the potential regulatory effect of these classical signaling events on mitochondrial function is of interest. However, the functional impact of protein phosphorylation and the kinase/phosphatase system responsible for it are relatively unknown. Exceptions include the well-characterized pyruvate dehydrogenase and branched chain ␣-ketoacid dehydrogenase regulatory system. The first task of this review is to update the current status of protein phosphorylation detection primarily in the matrix and evaluate evidence linking these events with enzymatic function or protein processing. To manage the scope of this effort, we have focused on the pathways involved in energy metabolism. The high sensitivity of modern methods of detecting protein phosphorylation and the low specificity of many kinases suggests that detection of protein phosphorylation sites without information on the mole fraction of phosphorylation is difficult to interpret, especially in metabolic enzymes, and is likely irrelevant to function. However, several systems including protein translocation, adenine nucleotide translocase, cytochrome c, and complex IV protein phosphorylation have been well correlated with enzymatic function along with the classical dehydrogenase systems. The second task is to review the current understanding of the kinase/phosphatase system within the matrix. Though it is clear that protein phosphorylation occurs within the matrix, based on 32 P incorporation and quantitative mass spectrometry measures, the kinase/phosphatase system responsible for this process is ill-defined. An argument is presented that remnants of the much more labile bacterial protein phosphoryl transfer system may be present in the matrix and that the evaluation of this possibility will require the application of approaches developed for bacterial cell signaling to the mitochondria.kinase; phosphatase; citric acid cycle; oxidative phosphorylation; protein transport; mitochondria intermembrane space; phosphohistidine; adenine nucleotide translocase; pyruvate dehydrogenase; branched chain ␣-ketoacid dehydrogenase THIS REVIEW ARTICLE is part of a collection on Post-translational Protein Modification in Metabolic Stress. Other articles appearing in this collection, as well as a full archive of all Review collections, can be found online at http://ajpheart. physiology.org/.