Effect of the mitochondrial membrane potential on the absorbance of the reduced form of cytochrome c oxidase.

The effect of mitochondrial membrane potential (ΔΨm) on the absorbance of the reduced cytochrome c oxidase (COX) was evaluated in isolated rabbit heart mitochondria using integrating sphere optical spectroscopy. Maximal reduction of the mitochondrial cytochromes was achieved by either blowing nitrogen to remove oxygen, or by adding cyanide. Gradual depolarization of ΔΨm by adding increasing concentrations of uncoupler resulted in an increase of up to 50 % in the absorbance of cytochrome aa3 under nitrogen saturation, and of 25 % with cyanide. Cytochrome aa3 absorbance increases were also observed in the presence of cyanide with apyrase (20 %) or oligomycin (12 %). The bL heme absorbance also decreased as expected from ΔΨm depolarization. A ~ 1 nm red shift in the peak wavelength of cytochrome aa3 was observed under anoxic conditions as ΔΨm was depolarized. Importantly, cytochrome c and c1 absorbances remained constant at levels corresponding to full reduction under all experimental manipulations of ΔΨm, especially with cyanide. These data suggest that ΔΨm-dependent changes in the absorbance of reduced COX were due to a variable extinction coefficient of heme a and/or a3 as a function of ΔΨm. A similar increase in the reduced cytochrome aa3 absorbance without changes in cytochrome c and c1 was observed in the perfused rabbit heart when decreasing ΔΨm with uncoupler. Our results imply that COX absorbance in its fully reduced state does not simply reflect the oxygen tension but also the ΔΨm. This may prove useful in monitoring ΔΨm under anoxic or ischemic conditions in intact tissue.

Lactate oxidation in Paracoccus denitrificans.

Paracoccus denitrificans has a classical cytochrome-dependent electron transport chain and two alternative oxidases. The classical transport chain is very similar to that in eukaryotic mitochondria. Thus, P. denitrificans can serve as a model of the mammalian mitochondrion that may be more tractable in elucidating mechanisms of regulation of energy production than are mitochondria. In a previous publication we reported detailed studies on respiration in P. denitrificans grown aerobically on glucose or malate. We noted that P. denitrificans has large stores of lactate under various growth conditions. This is surprising because P. denitrificans lacks an NAD+-dependent lactate dehydrogenase. The aim of this study was to investigate the mechanisms of lactate oxidation in P. denitrificans. We found that the bacterium grows well on either d-lactate or l-lactate. Growth on lactate supported a rate of maximum respiration that was equal to that of cells grown on glucose or malate. We report proteomic, metabolomic, and biochemical studies that establish that the metabolism of lactate by P. denitrificans is mediated by two non-NAD+-dependent lactate dehydrogenases. One prefers d-lactate over l-lactate (D-iLDH) and the other prefers l-lactate (L-iLDH). We cloned and produced the D-iLDH and characterized it. The Km for d-lactate was 34 µM, and for l-lactate it was 3.7 mM. Pyruvate was not a substrate, rendering the reaction unidirectional with lactate being converted to pyruvate for entry into the TCA cycle. The intracellular lactate was ∼14 mM such that both isomers could be metabolized by the enzyme. The enzyme has 1 FAD per molecule and utilizes a quinone rather than NAD + as an electron acceptor. D-iLDH provides a direct entry of lactate reducing equivalents into the cytochrome chain, potentially explaining the high respiratory capacity of P. denitrificans in the presence of lactate.

Cardiac nitric oxide scavenging: role of myoglobin and mitochondria.

Vascular production of nitric oxide (NO) regulates vascular tone. However, highly permeable NO entering the cardiomyocyte would profoundly impact metabolism and signalling without scavenging mechanisms. The purpose of this study was to establish mechanisms of cardiac NO scavenging. Quantitative optical studies of normoxic working hearts demonstrated that micromolar NO concentrations did not alter mitochondria redox state or respiration despite detecting NO oxidation of oxymyoglobin to metmyoglobin. These data are consistent with proposals that the myoglobin/myoglobin reductase (Mb/MbR) system is the major NO scavenging site. However, kinetic studies in intact hearts reveal a minor role (∼9%) for the Mb/MbR system in NO scavenging. In vitro, oxygenated mitochondria studies confirm that micromolar concentrations of NO bind cytochrome oxidase (COX) and inhibit respiration. Mitochondria had a very high capacity for NO scavenging, importantly, independent of NO binding to COX. NO is also known to quickly react with reactive oxygen species (ROS) in vitro. Stimulation of NO scavenging with antimycin and its inhibition by substrate depletion are consistent with NO interacting with ROS generated in Complex I or III under aerobic conditions. Extrapolating these in vitro data to the intact heart supports the hypothesis that mitochondria are a major site of cardiac NO scavenging. KEY POINTS: Cardiomyocyte scavenging of vascular nitric oxide (NO) is critical in maintaining normal cardiac function. Myoglobin redox cycling via myoglobin reductase has been proposed as a major NO scavenging site in the heart. Non-invasive optical spectroscopy was used to monitor the effect of NO on mitochondria and myoglobin redox state in intact beating heart and isolated mitochondria. These non-invasive studies reveal myoglobin/myoglobin reductase plays a minor role in cardiac NO scavenging. A high capacity for NO scavenging by heart mitochondria was demonstrated, independent of cytochrome oxidase binding but dependent on oxygen and high redox potentials consistent with generation of reactive oxygen species.

Spectroscopic identification of the catalytic intermediates of cytochrome c oxidase in respiring heart mitochondria.

The catalytic cycle of cytochrome c oxidase (COX) couples the reduction of oxygen to the translocation of protons across the inner mitochondrial membrane and involves several intermediate states of the heme a3-CuB binuclear center with distinct absorbance properties. The absorbance maximum close to 605 nm observed during respiration is commonly assigned to the fully reduced species of hemes a or a3 (R). However, by analyzing the absorbance of isolated enzyme and mitochondria in the Soret (420-450 nm), alpha (560-630 nm) and red (630-700 nm) spectral regions, we demonstrate that the Peroxy (P) and Ferryl (F) intermediates of the binuclear center are observed during respiration, while the R form is only detectable under nearly anoxic conditions in which electrons also accumulate in the higher extinction coefficient low spin a heme. This implies that a large fraction of COX (>50 %) is active, in contrast with assumptions that assign spectral changes only to R and/or reduced heme a. The concentration dependence of the COX chromophores and reduced c-type cytochromes on the transmembrane potential (ΔΨm) was determined in isolated mitochondria during substrate or apyrase titration to hydrolyze ATP. The cytochrome c-type redox levels indicated that soluble cytochrome c is out of equilibrium with respect to both Complex III and COX. Thermodynamic analyses confirmed that reactions involving the chromophores we assign as the P and F species of COX are ΔΨm-dependent, out of equilibrium, and therefore much slower than the ΔΨm-insensitive oxidation of the R intermediate, which is undetectable due to rapid oxygen binding.

Multiomics analyses reveal early metabolic imbalance and mitochondrial stress in neonatal photoreceptors leading to cell death in Pde6brd1/rd1 mouse model of retinal degeneration.

Retinal diseases exhibit extensive genetic heterogeneity and complex etiology with varying onset and severity. Mutations in over 200 genes can lead to photoreceptor dysfunction and/or cell death in retinal neurodegeneration. To deduce molecular pathways that initiate and/or drive cell death, we adopted a temporal multiomics approach and examined molecular and cellular events in newborn and developing photoreceptors before the onset of degeneration in a widely-used Pde6brd1/rd1 (rd1) mouse, a model of autosomal recessive retinitis pigmentosa caused by PDE6B mutations. Transcriptome profiling of neonatal and developing rods from the rd1 retina revealed early downregulation of genes associated with anabolic pathways and energy metabolism. Quantitative proteomics of rd1 retina showed early changes in calcium signaling and oxidative phosphorylation, with specific partial bypass of complex I electron transfer, which precede the onset of cell death. Concurrently, we detected alterations in central carbon metabolism, including dysregulation of components associated with glycolysis, pentose phosphate and purine biosynthesis. Ex vivo assays of oxygen consumption and transmission electron microscopy validated early and progressive mitochondrial stress and abnormalities in mitochondrial structure and function of rd1 rods. These data uncover mitochondrial overactivation and related metabolic alterations as determinants of early pathology and implicate aberrant calcium signaling as an initiator of higher mitochondrial stress. Our studies thus provide a mechanistic framework with mitochondrial damage and metabolic disruptions as early drivers of photoreceptor cell death in retinal degeneration.

Energy homeostasis is a conserved process: Evidence from Paracoccus denitrificans' response to acute changes in energy demand.

Paracoccus denitrificans is a model organism for the study of oxidative phosphorylation. We demonstrate a very high respiratory capacity compared to mitochondria when normalizing to cytochrome aa3 content even in the absence of alternative terminal oxidases. To gain insight into conserved mechanisms of energy homeostasis, we characterized the metabolic response to K+ reintroduction. A rapid 3-4-fold increase in respiration occurred before substantial cellular K+ accumulation followed by a sustained increase of up to 6-fold that persisted after net K+ uptake stopped. Proton motive force (Δp) was slightly higher upon addition of K+ with ΔpH increasing and compensating for membrane potential (ΔΨ) depolarization. Blocking the F0F1-ATP synthase (Complex V) with venturicidin revealed that the initial K+-dependent respiratory activation was primarily due to K+ influx. However, the ability to sustain an increased respiration rate was partially dependent on Complex V activity. The 6-fold stimulation of respiration by K+ resulted in a small net reduction of most cytochromes, different from the pattern observed with chemical uncoupling and consistent with balanced input and utilization of reducing equivalents. Metabolomics showed increases in glycolytic and TCA cycle intermediates together with a decrease in basic amino acids, suggesting an increased nitrogen mobilization upon K+ replenishment. ATP and GTP concentrations increased after K+ addition, indicating a net increase in cellular potential energy. Thus, K+ stimulates energy generation and utilization resulting in an almost constant Δp and increased high-energy phosphates during large acute and steady state changes in respiration. The specific energy consuming processes and signaling events associated with this simultaneous activation of work and metabolism in P. denitrificans remain unknown. Nevertheless, this homeostatic behavior is very similar to that observed in mitochondria in tissues when cellular energy requirements increase. We conclude that the regulation of energy generation and utilization to maintain homeostasis is conserved across the prokaryote/eukaryote boundary.

Genome-wide Profiling Identifies DNA Methylation Signatures of Aging in Rod Photoreceptors Associated with Alterations in Energy Metabolism.

Aging-associated functional decline is accompanied by alterations in the epigenome. To explore DNA modifications that could influence visual function with age, we perform whole-genome bisulfite sequencing of purified mouse rod photoreceptors at four ages and identify 2,054 differentially methylated regions (DMRs). We detect many DMRs during early stages of aging and in rod regulatory regions, and some of these cluster at chromosomal hotspots, especially on chromosome 10, which includes a longevity interactome. Integration of methylome to age-related transcriptome changes, chromatin signatures, and first-order protein-protein interactions uncover an enrichment of DMRs in altered pathways that are associated with rod function, aging, and energy metabolism. In concordance, we detect reduced basal mitochondrial respiration and increased fatty acid dependency with retinal age in ex vivo assays. Our study reveals age-dependent genomic and chromatin features susceptible to DNA methylation changes in rod photoreceptors and identifies a link between DNA methylation and energy metabolism in aging.

Perfused murine heart optical transmission spectroscopy using optical catheter and integrating sphere: Effects of ischemia/reperfusion.

Tissue transmission optical absorption spectroscopy provides dynamic information on metabolism and function. Murine genetic malleability makes it a major model for heart research. The diminutive size of the mouse heart makes optical transmission studies challenging. Using a perfused murine heart center mounted in an integrating sphere for light collection with a ventricular cavity optical catheter as an internal light source provided an effective method of optical data collection in this model. This approach provided high signal to noise optical spectra which when fit with model spectra provided information on tissue oxygenation and redox state. This technique was applied to the study of cardiac ischemia and ischemia reperfusion which generates extreme heart motion, especially during the ischemic contracture. The integrating sphere reduced motion artifacts associated with a fixed optical pickup and methods were developed to compensate for changes in tissue thickness. During ischemia, rapid decreases in myoglobin oxygenation occurred along with increases in cytochrome reduction levels. Surprisingly, when ischemic contracture occurred, myoglobin remained fully deoxygenated, while the cytochromes became more reduced consistent with a further, and critical, reduction of mitochondrial oxygen tension during ischemic contraction. This optical arrangement is an effective method of monitoring murine heart metabolism.

Paradoxical arteriole constriction compromises cytosolic and mitochondrial oxygen delivery in the isolated saline-perfused heart.

The isolated saline-perfused heart is used extensively to study cardiac physiology. Previous isolated heart studies have demonstrated lower tissue oxygenation compared with in vivo hearts based on myoglobin oxygenation and the mitochondrial redox state. These data, consistent with small anoxic regions, suggest that the homeostatic balance between work and oxygen delivery is impaired. We hypothesized that these anoxic regions are caused by inadequate local perfusion due to a paradoxical arteriole constriction generated by a disrupted vasoregulatory network. We tested this hypothesis by applying two exogenous vasodilatory agents, adenosine and cromakalim, to relax vascular tone in an isolated, saline-perfused, working rabbit heart. Oxygenation was monitored using differential optical transmission spectroscopy and full spectral fitting. Increases in coronary flow over control with adenosine (27 ± 4 ml/min) or cromakalim (44 ± 4 ml/min) were associated with proportional spectral changes indicative of myoglobin oxygenation and cytochrome oxidase (COX) oxidation, consistent with a decrease in tissue anoxia. Quantitatively, adenosine decreased deoxymyoglobin optical density (OD) across the wall by 0.053 ± 0.008 OD, whereas the reduced form of COX was decreased by 0.039 ± 0.005 OD. Cromakalim was more potent, decreasing deoxymyoglobin and reducing the level of COX by 0.070 ± 0.019 OD and 0.062 ± 0.019 OD, respectively. These effects were not species specific, as Langendorff-perfused mouse hearts treated with adenosine demonstrated similar changes. These data are consistent with paradoxical arteriole constriction as a major source of regional anoxia during saline heart perfusion. We suggest that the vasoregulatory network is disrupted by the washout of interstitial vasoactive metabolites in vitro. NEW & NOTEWORTHY Regional tissue anoxia is a common finding in the ubiquitous saline-perfused heart but is not found in vivo. Noninvasive optical techniques confirmed the presence of regional anoxia under control conditions and demonstrated that anoxia is diminished using exogenous vasodilators. These data are consistent with active arteriole constriction, occurring despite regional anoxia, generated by a disrupted vasoregulatory network. Washout of interstitial vasoactive metabolites may contribute to the disruption of normal vasoregulatory processes in vitro.

Cardiac performance is limited by oxygen delivery to the mitochondria in the crystalloid-perfused working heart.

The left ventricular working, crystalloid-perfused heart is used extensively to evaluate basic cardiac function, pathophysiology, and pharmacology. Crystalloid-perfused hearts may be limited by oxygen delivery, as adding oxygen carriers increases myoglobin oxygenation and improves myocardial function. However, whether decreased myoglobin oxygen saturation impacts oxidative phosphorylation (OxPhos) is unresolved, since myoglobin has a much lower affinity for oxygen than cytochrome c oxidase (COX). In the present study, a laboratory-based synthesis of an affordable perfluorocarbon (PFC) emulsion was developed to increase perfusate oxygen carrying capacity without impeding optical absorbance assessments. In left ventricular working hearts, along with conventional measurements of cardiac function and metabolic rate, myoglobin oxygenation and cytochrome redox state were monitored using a novel transmural illumination approach. Hearts were perfused with Krebs-Henseleit (KH) or KH supplemented with PFC, increasing perfusate oxygen carrying capacity by 3.6-fold. In KH-perfused hearts, myoglobin was deoxygenated, consistent with cytoplasmic hypoxia, and the mitochondrial cytochromes, including COX, exhibited a high reduction state, consistent with OxPhos hypoxia. PFC perfusate increased aortic output from 76 ± 6 to 142 ± 4 ml/min and increased oxygen consumption while also increasing myoglobin oxygenation and oxidizing the mitochondrial cytochromes. These results are consistent with limited delivery of oxygen to OxPhos resulting in an adapted lower cardiac performance with KH. Consistent with this, PFCs increased myocardial oxygenation, and cardiac work was higher over a wider range of perfusate Po2. In summary, heart mitochondria are limited by oxygen delivery with KH; supplementation of KH with PFC reverses mitochondrial hypoxia and improves cardiac performance, creating a more physiological tissue oxygen delivery. NEW & NOTEWORTHY Optical absorbance spectroscopy of intrinsic chromophores reveals that the commonly used crystalloid-perfused working heart is oxygen limited for oxidative phosphorylation and associated cardiac work. Oxygen-carrying perfluorocarbons increase myocardial oxygen delivery and improve cardiac function, providing a more physiological mitochondrial redox state and emphasizing cardiac work is modulated by myocardial oxygen delivery.

Intracardiac light catheter for rapid scanning transmural absorbance spectroscopy of perfused myocardium: measurement of myoglobin oxygenation and mitochondria redox state.

Absorbance spectroscopy of intrinsic cardiac chromophores provides nondestructive assessment of cytosolic oxygenation and mitochondria redox state. Isolated perfused heart spectroscopy is usually conducted by collecting reflected light from the heart surface, which represents a combination of surface scattering events and light that traversed portions of the myocardium. Reflectance spectroscopy with complex surface scattering effects in the beating heart leads to difficulty in quantitating chromophore absorbance. In this study, surface scattering was minimized and transmural path length optimized by placing a light source within the left ventricular chamber while monitoring transmurally transmitted light at the epicardial surface. The custom-designed intrachamber light catheter was a flexible coaxial cable (2.42-Fr) terminated with an encapsulated side-firing LED of 1.8 × 0.8 mm, altogether similar in size to a Millar pressure catheter. The LED catheter had minimal impact on aortic flow and heart rate in Langendorff perfusion and did not impact stability of the left ventricule of the working heart. Changes in transmural absorbance spectra were deconvoluted using a library of chromophore reference spectra to quantify the relative contribution of specific chromophores to the changes in measured absorbance. This broad-band spectral deconvolution approach eliminated errors that may result from simple dual-wavelength absorbance intensity. The myoglobin oxygenation level was only 82.2 ± 3.0%, whereas cytochrome c and cytochrome a + a3 were 13.3 ± 1.4% and 12.6 ± 2.2% reduced, respectively, in the Langendorff-perfused heart. The intracardiac illumination strategy permits transmural optical absorbance spectroscopy in perfused hearts, which provides a noninvasive real-time monitor of cytosolic oxygenation and mitochondria redox state.NEW & NOTEWORTHY Here, a novel nondestructive real-time approach for monitoring intrinsic indicators of cardiac metabolism and oxygenation is described using a catheter-based transillumination of the left ventricular free wall together with complete spectral analysis of transmitted light. This approach is a significant improvement in the quality of cardiac optical absorbance spectroscopic metabolic analyses.

Cytochrome c Oxidase Activity Is a Metabolic Checkpoint that Regulates Cell Fate Decisions During T Cell Activation and Differentiation.

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.

Quantification of Oxygen Consumption in Retina Ex Vivo Demonstrates Limited Reserve Capacity of Photoreceptor Mitochondria.

PURPOSE: Cell death in neurodegeneration occurs at the convergence of diverse metabolic pathways. In the retina, a common underlying mechanism involves mitochondrial dysfunction since photoreceptor homeostasis and survival are highly susceptible to altered aerobic energy metabolism. We sought to develop an assay to directly measure oxygen consumption in intact retina with the goal of identifying alterations in respiration during photoreceptor dysfunction and degeneration. METHODS: Circular punches of freshly isolated mouse retina, adjacent to the optic nerve head, were used in the microplate-based Seahorse Extracellular Flux Analyzer to measure oxygen consumption. Tissue integrity was evaluated by propidium iodide staining and live imaging. Different substrates were tested for mitochondrial respiration. Basal and maximal respiration were expressed as oxygen consumption rate (OCR) and respectively measured in Ames' medium before and after the addition of mitochondrial uncoupler, BAM15. RESULTS: We show that glucose is an essential substrate for retinal mitochondria. At baseline, mitochondria respiration in the intact wild-type retina was close to maximal, with limited reserve capacity. Similar OCR and limited mitochondrial reserve capacity was also observed in cone-only Nrl-/- retina. However, the retina of Pde6brd1/rd1, Cep290rd16/rd16 and Rpgrip1-/- mice, all with dysfunctional or no photoreceptors, had reduced OCR and higher mitochondrial reserve capacity. CONCLUSIONS: 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.

Stimulation of oxidative phosphorylation by calcium in cardiac mitochondria is not influenced by cAMP and PKA activity.

Cardiac oxidative ATP generation is finely tuned to match several-fold increases in energy demand. Calcium has been proposed to play a role in the activation of ATP production via PKA phosphorylation in response to intramitochondrial cAMP generation. We evaluated the effect of cAMP, its membrane permeable analogs (dibutyryl-cAMP, 8-bromo-cAMP), and the PKA inhibitor H89 on respiration of isolated pig heart mitochondria. cAMP analogs did not stimulate State 3 respiration of Ca2 +-depleted mitochondria (82.2 ± 3.6% of control), in contrast to the 2-fold activation induced by 0.95 µM free Ca2 +, which was unaffected by H89. Using fluorescence and integrating sphere spectroscopy, we determined that Ca2 + increased the reduction of NADH (8%), and of cytochromes bH (3%), c1 (3%), c (4%), and a (2%), together with a doubling of conductances for Complex I + III and Complex IV. None of these changes were induced by cAMP analogs nor abolished by H89. In Ca2 +-undepleted mitochondria, we observed only slight changes in State 3 respiration rates upon addition of 50 µM cAMP (85 ± 9.9%), dibutyryl-cAMP (80.1 ± 5.2%), 8-bromo-cAMP (88.6 ± 3.3%), or 1 µM H89 (89.7 ± 19.9%) with respect to controls. Similar results were obtained when measuring respiration in heart homogenates. Addition of exogenous PKA with dibutyryl-cAMP or the constitutively active catalytic subunit of PKA to isolated mitochondria decreased State 3 respiration by only 5­15%. These functional studies suggest that alterations in mitochondrial cAMP and PKA activity do not contribute significantly to the acute Ca2 + stimulation of oxidative phosphorylation

Optical spectroscopy in turbid media using an integrating sphere: mitochondrial chromophore analysis during metabolic transitions.

Recent evidence suggests that the activity of mitochondrial oxidative phosphorylation complexes (MOPCs) is modulated at multiple sites. Here, a method of optically monitoring electron distribution within and between MOPCs is described using a center-mounted sample in an integrating sphere (to minimize scattering effects) with a rapid-scanning spectrometer. The redox-sensitive MOPC absorbances (∼465-630 nm) were modeled using linear least squares analysis with individual chromophore spectra. Classical mitochondrial activity transitions (e.g., ADP-induced increase in oxygen consumption) were used to characterize this approach. Most notable in these studies was the observation that intermediates of the catalytic cycle of cytochrome oxidase are dynamically modulated with metabolic state. The MOPC redox state, along with measurements of oxygen consumption and mitochondrial membrane potential, was used to evaluate the conductances of different sections of the electron transport chain. This analysis then was applied to mitochondria isolated from rabbit hearts subjected to ischemia/reperfusion (I/R). Surprisingly, I/R resulted in an inhibition of all measured MOPC conductances, suggesting a coordinated down-regulation of mitochondrial activity with this well-established cardiac perturbation.

Continuous monitoring of enzymatic activity within native electrophoresis gels: application to mitochondrial oxidative phosphorylation complexes.

Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light-scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze the enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction medium recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high-resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase in which catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed.

Cardiac mitochondrial matrix and respiratory complex protein phosphorylation.

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 α-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.

Regulation of oxidative phosphorylation complex activity: effects of tissue-specific metabolic stress within an allometric series and acute changes in workload.

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.

Homogenous protein programming in the mammalian left and right ventricle free walls.

Despite identical cardiac outputs, the right (RV) and left ventricle (LV) have very different embryological origins and resting workload. These differences suggest that the ventricles have different protein programming with regard to energy metabolism and contractile elements. The objective of this study was to determine the relative RV and LV protein expression levels, with an emphasis on energy metabolism. The RV and LV protein contents of the rabbit and porcine heart were determined with quantitative gel electrophoresis (2D-DIGE), mass spectrometry, and optical spectroscopy techniques. Surprisingly, the expression levels for more than 600 RV and LV proteins detected were similar. This included proteins many different compartments and metabolic pathways. In addition, no isoelectric shifts were detected in 2D-DIGE consistent with no differential posttranslational modifications in these proteins. Analysis of the RV and LV metabolic response to work revealed that the metabolic rate increases much faster with workload in the RV compared with LV. This implies that the generally lower metabolic stress of the RV actually approaches LV metabolic stress at maximum workloads. Thus, identical levels of energy conversion and mechanical elements in the RV and LV may be driven by the performance requirements at maximum workloads. In summary, the ventricles of the heart manage the differences in overall workload by modifying the amounts of cytosol, not its composition. The constant myocyte composition in the LV and RV implies that the ratio of energy metabolism and contractile elements may be optimal for the sustained cardiac contractile activity in the mammalian heart.

Intrinsic protein kinase activity in mitochondrial oxidative phosphorylation complexes.

Mitochondrial protein phosphorylation is a well-recognized metabolic control mechanism, with the classical example of pyruvate dehydrogenase (PDH) regulation by specific kinases and phosphatases of bacterial origin. However, despite the growing number of reported mitochondrial phosphoproteins, the identity of the protein kinases mediating these phosphorylation events remains largely unknown. The detection of mitochondrial protein kinases is complicated by the low concentration of kinase relative to that of the target protein, the lack of specific antibodies, and contamination from associated, but nonmatrix, proteins. In this study, we use blue native gel electrophoresis (BN-PAGE) to isolate rat and porcine heart mitochondrial complexes for screening of protein kinase activity. To detect kinase activity, one-dimensional BN-PAGE gels were exposed to [γ-(32)P]ATP and then followed by sodium dodecyl sulfate gel electrophoresis. Dozens of mitochondrial proteins were labeled with (32)P in this setting, including all five complexes of oxidative phosphorylation and several citric acid cycle enzymes. The nearly ubiquitous (32)P protein labeling demonstrates protein kinase activity within each mitochondrial protein complex. The validity of this two-dimensional BN-PAGE method was demonstrated by detecting the known PDH kinases and phosphatases within the PDH complex band using Western blots and mass spectrometry. Surprisingly, these same approaches detected only a few additional conventional protein kinases, suggesting a major role for autophosphorylation in mitochondrial proteins. Studies on purified Complex V and creatine kinase confirmed that these proteins undergo autophosphorylation and, to a lesser degree, tenacious (32)P-metabolite association. In-gel Complex IV activity was shown to be inhibited by ATP, and partially reversed by phosphatase activity, consistent with an inhibitory role for protein phosphorylation in this complex. Collectively, this study proposes that many of the mitochondrial complexes contain an autophosphorylation mechanism, which may play a functional role in the regulation of these multiprotein units.