Participation of Free Radicals in Photoreduction of Protochlorophyllide to Chlorophyllide in an Artificial Pigment-Protein Complex

O. B. Belyaeva^1*, W. T. Griffiths², J. V. Kovalev¹, K. N. Timofeev¹, and F. F. Litvin¹

¹School of Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-4309; E-mail: olgabelyaeva@mail.ru

²Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK

^* To whom correspondence should be addressed.

Received March 16, 2000; Revision received September 25, 2000

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The primary stages of protochlorophyllide phototransformation in an artificially formed complex containing heterologously expressedphotoenzyme 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.

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KEY WORDS: protochlorophyllide, protochlorophyllide-oxidoreductase, non-fluorescence intermediate, ESR signal

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