Coupled electron and proton transfer is recognized as the universal principle of primary energy conservation in respiration. Terminal respiratory oxidases are membrane-bound electron- transfer complexes that catalyze the reduction of oxygen to water and this reaction is associated with the generation of a transmembrane proton gradient that is the primary source of cellular free energy. Oxidases utilize two basic principles to produce the proton gradient. The first is a classical Mitchell's loop mechanism in which electrons and protons for the water formation are taken up from the opposite sides of membrane. The second one is proton pumping in which proton transfer across the membrane is driven by the free energy provided by a redox reaction. However, the mechanism of proton pumping has not been elucidated in any proton pump driven by reduction-oxidation reactions and it remains one of the key problems of molecular bioenergetics. Proposed investigations will contribute to an understanding of the proton translocation mechanism by identification of the electron-proton coupling sites and experimental examination and establishment of the mechanistic principles of pumping and proton gating in heme-copper oxidases. The application of optical spectroscopy, electron paramagnetic resonance, rapid freeze-quenching, flash photolysis, stopped-flow, and several biochemical methods will be utilized to achieve these goals.
Specific aims will be addressed using the purified bovine heart oxidase, enzyme incorporated into phospholipid vesicles and in submitochondrial particles.
The investigation of molecular mechanisms of respiration constitutes a basic component of broader mitochondrial research, which is receiving increased interest due to the recognition that mitochondrial defects are linked to a wide range of degenerative diseases and free-radical generation, both of which can lead to premature aging and cancer. Mitochondria are also implicated in apoptosis, and attention has recently been drawn to the physiologically important action of the signaling molecule nitric oxide in inhibiting the respiration. Completion of this project will contribute to understanding of coupling electron transport to proton pumping and ATP synthesis in mitochondria and to the pathophysiology of various mitochondrial diseases.
