Tyr275 and Lys279 stabilize NADPH within the catalytic site of NADPH : protochlorophyllide oxidoreductase and are involved in the formation of the enzyme photoactive state

Fluorescence spectroscopic and kinetic analysis of photochemical activity, cofactor and substrate binding, and enzyme denaturation studies were perfor med with highly purified, recombinant pea NADPH: protochlorophyllide oxidor eductase (POR) heterologously expressed in Escherichia coli. The results ob tained with an individual stereoisomer of the substrate [C8-ethyl-C13(2)-(R )-protoclilorophyllide] demonstrate that the enzyme photoactive state posse sses a characteristic fluorescence maximum at 646 nm that is due to the pre sence of specific charged amino acids in the enzyme catalytic site. The pho toactive state is converted directly into an intermediate having fluorescen ce at 685 nm in a reaction involving direct hydrogen transfer from the cofa ctor (NADPH). Site-directed mutagenesis of the highly conserved Tyr275 (Y27 5F) and Lys279 (K279I and K279R) residues in the enzyme catalytic pocket de monstrated that the presence of these two amino acids in the wild-type POR considerably increases the probability of photoactive state formation follo wing cofactor and substrate binding by the enzyme. At the same time, the pr esence of these two amino acids destabilizes POR and increases the rate of enzyme denaturation. Neither Tyr275 nor Lys279 plays a crucial role in the binding of the substrate or cofactor by the enzyme. In addition, the presen ce of Tyr275 is absolutely necessary for the second step of the protochloro phyllide reduction reaction, "dark" conversion of the 685 nm fluorescence i ntermediate and the formation of the final product, chlorophyllide. We prop ose that Tyr275 and Lys279 participate in the proper coordination of NADPH and PChlide in the enzyme catalytic site and thereby control the efficiency of the formation of the POR photoactive state.