Dynamic Processes in Metalloprotein Complexes

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KosticMCB 9808392A. technical The long-term goal of this research is to explain, at the molecular level, the mechanism of electron-transfer reactions between metalloproteins and the importance of protein-protein docking and of dynamic rearrangement processes for these reactions. This understanding is important because metalloproteins and their complexes, as electron carriers and redox enzymes, are essential to metabolism. The objective is to understand fundamental principles of biological reactivity, specifically with proteins that are physiological partners. Instead of kinetics of biological (thermal) reactions, the focus is to use kinetics of closely related abiological (photoinduced) redox reactions as a tool to elucidate dynamic processes that modulate the overall biological reactions. These dynamic processes, however, may be masked in the thermal reactions. In the photoinduced electron transfer, which is much faster, the processes of interest can be decoupled from the masking processes and investigated quantitatively. The study is rooted in P.I.'s past accomplishments, but strikes in new directions. Recent determinations of the three-dimensional structures of cytochrome f, plastocyanin, and cytochrome c6 from the green algae Chlamydomonas reinhardtii and successes by collaborators at Berkeley and UCLA in molecular biology set the stage for these biophysical and biochemical investigations. The plan of work combines applied molecular biology (site-directed mutagenesis), kinetics of photoinduced redox reactions (laser flash photolysis), and spectroscopy (NMR and optical absorption and emission) in order to answer the following questions. (1) What dynamic processes possibly modulate the reaction of cytochrome f with its physiologically- equivalent oxidants, plastocyanin and cytochrome c6? (2) How does the remarkable two-domain structure of cytochrome f control the recognition and the reaction between this protein and its physiological partners. (3) Does cytochrome c6 contain a residue functionally equivalent to Tyr 83 in plastocyanin? (4) Does cytochrome c6 use its acidic patch in the reaction with cytochrome f ? Recognition patches on the protein surfaces will be altered by structurally-noninvasive mutations. Kinetic effects of these mutations and of ionic strength will reveal much about the protein-protein association. If flexible diprotein complexes rearrange from the initial, docking configuration to a different, reactive configuration, quantitative analysis of the effects of solution viscosity and of temperature will reveal the nature of this rearrangement and its trajectory. Some of the aforementioned issues are being debated, and others have barely been touched by previous researchers. The questions nos. 3 and 4 arose from very recent theoretical analyses, and all of the answers will spur further theoretical work. This project will stimulate thinking and rethinking about structure-function relationships for recognition and electron transfer in biological systems.2. Non-technicalAt the most elementary level, processes of life are chemicalprocesses. Therefore, experimental and theoretical methods of chemistry can reveal fundamental interactions and transformations involving biological molecules, such as proteins. Electrons are fundamental constituents of all atoms and the smallest particles carrying negative electrical charge. Flow of electrons in the living cell is an essential process for all organisms that carry out photosynthesis. Because all forms of life are interdependent, electron flow in cells is an essential requirement for life. Electron carriers in cells are metalloproteins - proteins containing metal atoms. This study deals with basic steps on the electron paths of three proteins: Cytochrome f, plastocyanin, and cytochrome c6, all from the same green alga, in their natural and mutated forms of these proteins and also with slightly altered forms. The speeds at which these proteins transfer the electrons between themselves are measured by laser methods. Experiments are designed to determine how the function of these proteins is governed by their three-dimensional structures and properties of the protein surfaces, and how purposeful alterations in the proteins affect the speed of transfer. Existing theoretical predictions will be tested and the results used to spur theorists to new analyses. This interplay between chemistry and molecular biology and between experiment and theory is advantageous in the study of fundamental principles of life.