Biological 02 production is the major renewable source of atmospheric oxygen on Earth. Water is the source of 02 created by photosynthesis in all contemporary photoautotrophic organisms and no other biological process is capable of splitting water into 02. The existence and health of all organisms that use aerobic metabolism, including humans, depends on photosynthetically derived 02. Catalysis of this chemistry is so complex that only a single type of enzymatic site appears to exist within all oxygenic photoautotrophs examined to date. The long term goal of this proposal is to reveal the chemical basis for biological water splitting. The approach to be used is to disassemble the labile inorganic core (Mn4OxCal CI1-2) comprising the active site of the enzyme and to reconstruct it from simple inorganic cofactors, atom-by-atom, using a controlled sequence of photo- and dark- reactions that is known already to fully restore the functional enzyme. This in vitro process is called photoactivation, and serves as a model for the in vivo biogenesis reactions. The assembly of """"""""inorganic mutants"""""""" of the active site by use of surrogate cofactors will be used to probe the function of the native cofactors. Information on the kinetics of assembly, the atomic composition/structure and the atomic forces will be acquired on the assembly intermediates and the intact holo-enzyme. Tools to be used include novel methods for detection of dissolved 02 concentration, proton concentration and a collection of electron??spin resonance techniques for time-resolved and multi-dimensional spectroscopy on paramagnetic systems (EPR-plus). Studies will focus on the enzyme from O2-producing plants and primitive cyanobacteria. The biomedical benefits of the proposed research should provide: 1) fundamental information about the structure of the water splitting enzyme and its mechanism of catalysis; 2) general understanding of the mechanism of assembly of metals into metalloproteins; 3) a molecular blueprint for design of a water oxidation catalyst based on the principles adopted by the photosynthetic enzymes; 4) general insights into how complex multi-metal enzymes cooperate in activation of substrates for catalysis of bond cleavage chemistry.
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