The cytochrome (cyt) bc1 complex plays a central role in respiratory and photosynthetic electron transport chains, but is also implicated in the production of damaging free radicals and reactive oxygen species relevant to pathogenesis and aging. In addition, the bc1 complex is the target of many biocidal drugs, with significant species specificity and range. Understanding the molecular mechanisms of the bc1 complex is therefore of major importance for both medical and basic research. Biochemical, biophysical and molecular biology studies have driven significant progress in the characterization of the structure and function of bc1 complexes, culminating in the X-ray structure of bc1 crystals from different sources, demonstrating that it is a homodimer. The proposed research focuses on the molecular mechanisms of bc1 activity, utilizing the kinetic and spectroscopic advantages of light activation of the complex from Rhodobacter sphaeroides, as well as its excellent molecular engineering attributes. The proposed research combines methods of high-throughput kinetic (single and multichannel) optical spectroscopy, chemometrics, infrared spectroscopy (FTIR), EPR and direct electric potential measurements, with the selective use of mutants in a histidine-tagged background. A novel dual affinity tag procedure is proposed for the generation of heterodimers with different mutations in each monomer.
Specific aims i nclude: (1) characterization of molecular mechanisms of coupling between electron transfer and protolytic reactions in the bc1 complex;(2) determination of the kinetics and equilibria between components as a function of transmembrane proton gradient, Ap, thereby identifying membrane potential (??) and pH gradient (?pH) sensitive steps;(3) characterization of changes in behavior of the bc1 complex as ??;builds up, such as cross-over ET between monomers, superoxide production, etc., using a calibrated carotenoid bandshift as voltmeter;(4) using a multi-pronged approach to demonstrate that electron transfer occurs between monomers;(5) testing our hypothesis that monomer-monomer electron transfer is efficient only under coupled conditions, and characterization the response of the monomer-monomer electron transfer to different factors, in particular ?pH, ??, and T;(6) determination of the energetics of the b-hemes and the Qj site quinone states. To achieve these goals, protocols have been devised for the specific isolation of many steps in the turnover of the bc1 complex, for study by multichannel spectroscopy, FTIR, and electrometric methods. These include: (i) Preparation of the system - reaction centers (RCs) and bc1 - in defined states by changing redox potential, pH and ionic strength, (ii) Separation of the RC and bc1 reactions on the basis of two-flash experiment (distinguishing donor and acceptor sides of the bc1 complex), (iii) Separation of reactions by removing electron-transport components (cyt c2, QB and other quinones) by traditional biochemical (extraction) methods, (iv) Isolation of different reactions by specific inhibitors of the bc1 complex and RC. (v) Deconvolution of spectral, kinetic and thermodynamic parameters, (vi) Use of selected mutants in a histidine-tagged environment to limit or eliminate specific reactions or states of the bc1 complex;(vii) Creation of a heterodimer of the bc1 complex by means of dual affinity tags. The relevance of this work to human health is significant. The cyt bc1 complex, or Complex III, is a major site of production of damaging oxidizing species that cause slow accumulation of damage to mitochondria, especially the DNA. This is thought to be a significant contributor to the aging process. Several chronic and congenital diseases (myopathies) also arise from malfunctioning bc1 complex. Finally, this activity is a target for new therapeutic agents, especially for fungi and parasitic protists.
