EXCEED THE SPACE PROVIDED. The cytochrome bc[ complex, which plays a central role in respiratory and photosynthetic electron transport chains, is also implicated in the production of damaging free radicals and reactive oxygen species relevant to pathogenesis and aging. Understanding the molecular mechanisms of the bci complex is therefore of major importance for medical and basic research. Biochemical, biophysical and molecular biology studies have driven significant progress in the characterization of the structure and function of b^ complexes, culminating in the X-ray structure of bC[ crystals from different sources. In spite of a broad knowledge- base on bC]complex function, our understanding of the molecular mechanisms governing its function is crude. With the molecular structure of the bc[ complex in hand we are now in a position to study the molecular mechanisms of the b^ complex, i.e., the sequence of time dependent and synchronous interactions between cofactors and protein at molecular and atomic levels. This proposal focuses on the molecular mechanisms of bci turnover, utilizingthe kinetic and spectroscopic advantages of light activation of the complex from Rhodobacter sphaeroides. The proposed research combines the powerful methods of infrared spectroscopy (FTIR), multichannel optical spectroscopy, chemometrics, and direct electric potential measurements, with the selective use of mutants in a histidine-tagged background.
Specific aims i nclude: (1) localization of protonogenic and electrogenic steps in cytochrome b^ turnover and determination of their relativecontributions to net electrogenesis; (2) molecular characterization of the protolytic reactions resulting in proton uptake and proton release; (3) application of FTIR to identify IR bands of the cofactors, and responses of the protein due to binding of specific inhibitors in centers b' and V of the be] complex; (4) identification by FTIR and mutagenesis of the principle components of the proton conduction pathways, especially amino acid residues; and (5) characterization of molecular mechanisms of coupling between electron transfer and protolytic reactions of the ubiquinonesin the bcj complex. To achieve these goals, protocols have been devised for the specific isolation of many steps in the turnover of the bcj complex, for study by FTIR, multichannel spectroscopy, and electrometric methods. These include: (i) Preparation of the system - reaction centers (RCs) and be, - in defined states by changing redox potential, pH and ionic strength, (ii)Separation of the RC and be! reactions on the basis of two-flash experiment (distinguishing donor and acceptor sides of the bci complex), (iii) Separation of reactions by removing electron-transport components (cytochrome c2, QBand other quinones) by traditional biochemical (extraction) methods, (iv) Isolation of different reactions by specific inhibitors of the bci complex and RC. (v) Deconvolution of spectral, kinetic and thermodynamicparameters, (vi) Use of selected mutants in a histidine-tagged environment to limit or eliminate specific reactions or states of the bC[complex.
