DISTANCE MEASUREMENTS IN METALLOPROTEINS

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DESCRIPTION: (Adapted from the Applicant's Abstract) Energy transduction in respiration and photosynthesis involves the coupling of electron-transfer reactions to the generation of an electrochemical gradient across a bilayer membrane. Although the sequence and kinetics of the electron-transfer reactions are by and large known in these systems, much less is known about the distances and electronic couplings between the electron carriers. The long term goals of this project are to develop and apply methods to use the electron carriers as probes to determine the distances and mechanisms of long-range biological electron transfer and the structure and magnetic properties of multinuclear metal ion clusters. The primary objective of this proposal is to use electron spin relaxation enhancement measurements and analysis of electron paramagnetic resonance (EPR) line shapes to obtain structural and magnetic information on photosystem II and nitric oxide synthase. The specific aims are to determine the spatial organization of the redox centers, the structure of the active site for water oxidation and the magnetic properties of the manganese cluster in photosystem II, and to characterize the conformational changes of nitric oxide synthase and its reductase domain upon activation by calmodulin. The next objectives are aimed at improving the methods for using spin relaxation enhancement measurements to determine distances. Photosystem I will be used as a structurally characterized model system to carry out a systematic study of the effects of distance, orientation, g-anisotropy, and spin distribution in a metal ion cluster on electron spin-lattice relaxation enhancement of the chlorophyll a special pair cation radical, P700+, by the reduced iron-sulfur clusters A, B, and X. This information is essential for accurate distance estimates between electron-transfer partners in redox proteins. In addition, the method of using endogenous Dy3+-chelate complexes as electron spin-relaxation enhancement agents to determine the surface accessibility of paramagnetic centers in proteins will be extended by using a Monte Carlo method to model the excluded protein volume and the distribution of Dy3+-chelate complexes in the solution surrounding the protein. Overall, these measurements will provide new information about the systems under study and will also provide a basis for using electron spin-lattice relaxation measurements to determine distances of electron transfer and magnetic properties of metal ion clusters in other redox protein complexes.