Participation of free radicals in photoreduction of protochlorophyllide to chlorophyllide in an artificial pigment-protein complex

The primary stages of protochlorophyllide phototransformation in an artificially formed complex containing heterologously expressed photoenzyme protochlorophyllide-oxidoreductase (POR), protochlorophyllide, and NADPH were investigated by optical and ESR spectroscopy. An ESR signal (g = 2.002; H = 1 mT) appeared after illumination of the complex with intense white light at 77 K. The ESR signal appeared with simultaneous quenching of the initial protochlorophyllide fluorescence, this being due to the formation of a primary non-fluorescent intermediate. The ESR signal disappeared on raising the temperature to 253 K, and a new fluorescence maximum at 695 nm belonging to chlorophyllide simultaneously appeared. The data show that the mechanism of protochlorophyllide photoreduction in the complex is practically identical to the in vivo mechanism: this includes the formation of a short-lived non-fluorescent free radical that is transformed into chlorophyllide in a dark reaction.