The ubiquinone pool and ubiquinol-cytochrome c oxidoreductase (cytochrome bc1 or Complex III) are central to cellular energy conversion in a very broad range of biological systems. Complex III converts oxidation- reduction (redox) free-energy stored in the substrates ubiquinol and cytochrome c into a transmembrane electric potential and pH gradient essential for cellular maintenance and, because of its reversibility, mitochondrial regulation. However, the Qo site of primary energy conversion in Complex III is poorly defined in crystal structures and experimentally difficult to access. We use molecular biology to excise all cofactors of the high and low potential chains except those that allow us to access to the molecular mechanisms of Complex III. These cofactor `knockouts'in the photosynthetic bacterium Rhodobacter capsulatus, are studied in vivo or in isolated reaction center/Complex III assemblies. The knockouts permit selective, controlled light generation of key catalytic states of reversible Qo site oxidation-reduction and coupled energy conversion. These quasi- equilibrium catalytic states are constrained to the Qo site, generated in high yields and persist for seconds at room temperatures. We expect in certain cases that they can be stabilized indefinitely. It is now viable to investigate these newly isolated catalytic states with a range of conventional and advanced structural, spectroscopic and electrochemical methods. These include infrared (ATR-FTIR) and electron paramagnetic resonance (EPR) to describe redox structures and mechanistic chemistry of the Qo site cofactors, quinone/quinol and recently, possibly the semiquinone radical in the quasi-equilibrium states. We hope to crystallize stabilized quasi-equilibrium Qo site states for X-ray structures of mechanistically relevant structures not considered possible before. Moreover, the lifetime of the quasi-equilibrium states lends each of them to be individually tested for reactivity with molecular oxygen with a view to identify mechanism and role in superoxide radicals and health critical ROS generation. All approaches promise novel molecular level descriptions of functional involvement of critical amino acids during active quinone-quinol binding and exchange, quinone- quinol oxidation-reduction and coupled charge separation catalysis. We believe that the approaches will also provide insight into the defensive strategies adopted by the Qo site for suppression of short circuits as well into the regulation of ROS. Experiments aided by general application of electron tunneling expressions will explore the underlying engineering of the Complex III, and other respiratory complexes for normal operation in forward and reverse modes, as well as determining the thresholds of failure and when destructive side reactions, including ROS generation, become significant. All aspects of the work will help bridge the significant gap between respiratory mechanism/energetics and mitochondrial and hence cellular regulation. PUBLIC HEALTH RELEVANCE The energy obtained by our food and breath is managed at the molecular level by a set of specific proteins that produce and control the high energy chemicals essential for life. We aim to understand how this molecular machinery controls such reactive chemicals as oxygen radicals to allow, on the one hand, normal healthy development and growth, and to minimize, on the other hand, the cellular damage that leads to aging and age-related diseases.