Although the chlorophyll biosynthetic pathway has been the subject of intense investigation over the past 50 years, much remains to be discovered. One such mystery involves the remarkable color change, from yellow to green, that occurs when dark-brown plants are transferred to the light. This """"""""greening reaction"""""""" involves the reduction of protochlorophyllide to chlorophyllide and is a key regulatory step, not alone in the biosynthesis of chlorophyll, but also in the development of the chloroplast. In this proposal model studies related to the mechanism of this important reaction are described. We propose that the reduction of protochlorophyllide may proceed via the protochlorophyllide radical cation or radical anion. To test the chemical plausibility of these pathways we will synthesize two model systems in which dihydronicotinamide is linked to tetra phenylporhpyrin and to protochlorophyllide. The feasibility of the radical cation pathway will be examined by oxidizing both model systems 'to the porphyrin radical cation and determining its reactivity with the appended dihydronicotinamide. The rate and the isotope effect on the hydride transfer will be determined. The radical anion pathway will be examined by direct irradiation of the model systems. Light dependent deuterium incorporation from the solvent into unreacted starting material will be examined as a simple probe for the porphyrin radical anion. The absorption spectra and the lifetimes of reaction intermediates will be determined. Isotope effects (solvent and dihydronicotinamide) on these lifetimes will be measured. The knowledge gained from these model studies on the properties of the intermediates involved in the reduction of protochlorophyllide by dihydronicotinamide will be used to interpret the transient absorption spectroscopy of the enzymatic reaction.
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