The purpose of these studies is to establish a better understanding of the energy metabolism of biological tissues using modern system biology approaches. Towards this goal, the laboratory concentrates on the use of screening approaches in proteomics, metabolomics, protein structure, post-translational modifications, minimally invasive metabolic rate information and optical spectroscopy. One of the major hypothesizes in this program is that the activity of the multi-protein Complexes that perform Oxidative Phosphorylation are coordinated in some fashion to balance the rate of ATP production with utilization in the cell. This results in the observed metabolic homeostasis where the potential energy for doing work is maintained near constant in the cell even during major alterations in workload. The following major findings were made over the last year: 1) Our previous work on the regulation of oxidative phosphorylation has concentrated on isolated mitochondria that we have extrapolated to in vivo conditions. Based on our realization that isolated mitochondria are structurally compromised when compared to the normal cellular structure, we have now moved our non-invasive optical studies of the chromophores of oxidative phosphorylation to evaluate the enzymatic activity of mitochondrial energy conversion to the study of the isolated working perfused heart. We have established a working rabbit heart preparation in the laboratory where we can simultaneously monitor heart work, metabolic rate and optically follow the redox state of all of the Complexes of oxidative phosphorylation, non-invasively. In addition to a white light LED tipped catheter previously reported, we have developed a side firing micro optical catheter capable of inserting in to the in vitro or in vivo mouse heart using an external light source. This development greatly expands the utility of this transmural optical spectroscopy experiment in the intact heart. 2) To broaden our analysis of metabolic regulation in the mitochondria we have expanded our studies to study the ancestors of mitochondria, simple bacteria. We have initiated studies on isolated bacteria believed to be closest to the mitochondrial origins, paracoccus denitrificans(PD). The goal of these studies is to unravel acute energy conversion regulation in this bacterium and then look for similar mechanisms in mammalian mitochondria. With the growing interest in the microbiome, these studies should also provide new insight into the acute regulation of bacterial energy metabolism that has not been extensively studied. This might provide new tools in generating novel antibiotics. In the last year we have begun a systems biology approach to studying the metabolic function of PD including the quantitation of the oxidative phosphorylation Complexes along with the absorption spectrum of the chromophores associated with these Complexes, protein and metabolite content (so called proteomics and metabolomics), ion transport, oxidative phosphorylation Complex redox state, oxygen consumption, fixed acid production, membrane potential and the NADH redox state. The acute work function of the bacteria we are exploring is volume regulation, that is the work associated with the bacterium adjusting to environmental salt content. These changes routinely occur in the soil or clinically in the gut and skin. One complication of these studies to define the energetics of this pre-mitochondria organism model is the monitoring of intracellular pH. Exogenous optical probes have proven unreliable thus two new approaches have been taken: A) genetically insert a pH sensitive green fluorescent protein into the PD genome or B) Develop a high performance 31P NMR chamber to monitor the chemical shift of intracellular PI to determine pH along with the phosphate metabolites involved in energetics such as ATP. Both of these projects have been initiated with promising initial results. 3) One hypothesis concerning the coordinated regulation of oxidative phosphorylation complexes is the formation of super complexes of these enzymes. In a super complex all of the complexes associated with generating the mitochondrial membrane potential, a primary energy source for the formation of ATP, are all co-localized in a large single unit that could modulate the function of all simultaneously via structural alterations of the Supercomplex. This is an attractive concept since simple modifications could result in alterations in several enzyme systems simultaneously. However, it is controversial whether these complexes actually exist in functioning mitochondria or whether they are an artifact of the detergents used to isolate the membrane bound proteins. To address this problem completed a collaboration with Dr. Heck in the Netherlands to examine whether the Complexes are structurally close to each other in intact mitochondria by using lipid permeable crosslinking agents in intact mitochondria for mass spectroscopy analysis. The existence of multi Complex Super-Complexes was confirmed in this analysis but an apparently much more extensive interaction of the Complexes were observed than previously appreciated. This was particularly true for Complex V usually ignored in these analysis. Many new proteins protein interactions were also discovered by this screening approach that will need to be confirmed with other methods and approaches. Clearly, the protein-protein interactome is extensive in the mitochondria. This approach also allowed us to separate the different compartments of the organelle including the matrix, interspace and different sides of the individual membranes. This should provide a novel insight into the compartmentation and interaction of the mitochondrial proteome. Current studies are focusing on this approach in intact PD cells to get the interactions as close as possible to the in vivo condition. Hopefully these studies will reveal the role of protein protein interactions in the regulation of mitochondria metabolism in the heart and many other tissues.
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