Protochlorophyllide oxidoreductase B-catalyzed protochlorophyllide photoreduction in vitro: Insight into the mechanism of chlorophyll formation in light-adapted plants

The mechanism of the protochlorophyllide (PChlide) photoreduction reaction operating in light-adapted plants and catalyzed by NADPH:protochlorophyllid e oxidoreductase B (PORb) has been analyzed by lore-temperature fluorescenc e spectroscopy by using purified barley PORb overexpressed heterologously i n Escherichia coli as a fusion protein with the maltose-binding protein. We show that the PORb-catalyzed PChlide reduction reaction consists of two st eps, one photochemical and the other nonphotochemical, The initial photoche mical reaction follows a single quantum mechanism and leads to the formatio n of an unstable intermediate with mixed pigment electronic structure and a n EPR spectrum that suggests the presence of a free electron. The second st ep involves the spontaneous conversion of the unstable intermediate into ch lorophyllide as defined by its spectroscopic characteristics anti migration on an HPLC column. Both steps of the reaction can be performed at subzero temperatures in frozen samples, suggesting that they do not include major c hanges in enzyme conformation or pigment rearrangement within the active si te. The rate of the reaction at room temperature depends linearly on enzyme and substrate (PChlide) concentration, and the kinetic parameters are cons istent with one molecule of substrate bound per active monomer in solution. The PORb-catalyzed reaction in vitro is spectroscopically similar to that identified in leaves of light-adapted plants, suggesting that the same reac tion sequence observed operates in planta.