tailieunhanh - Báo cáo khoa học: Dynamics of flavin semiquinone protolysis in L-a-hydroxyacid-oxidizing flavoenzymes – a study using nanosecond laser flash photolysis

The reactions of the flavin semiquinone generated by laser-induced stepwise two-photon excitation of reduced flavin have been studied previously (El Hanine-Lmoumene C & Lindqvist L. (1997) Photochem Photobiol66, 591–595) using time-resolved spectroscopy. In the present work, we have used the same experimental procedure to study the flavin semiquinone in rat kidney long-chain hydroxy acid oxidase and in the flavodehydrogenase domain of flavocytochrome b2 FDH | ễFEBS Journal Dynamics of flavin semiquinone protolysis in L-a-hydroxyacid-oxidizing flavoenzymes - a study using nanosecond laser flash photolysis Lars Lindqvist1 Simona Apostol1 Chaibia El Hanine-Lmoumene1 and Florence Lederer2 1 Laboratoire de Photophysique Moleculaire du Centre Nationalde la Recherche Scientifique Universite Paris-Sud 91405 Orsay France 2 Laboratoire de Chimie Physique Centre Nationalde la Recherche Scientifique UMR 8000 Universite Paris-Sud 91405 Orsay France Keywords flavin semiquinone flavocytochrome b2 long-chain hydroxy acid oxidase nanosecond laser photolysis proton transfer kinetics Correspondence L. Lindqvist Laboratoire de Photophysique Moleculaire du CNRS Universite Paris-Sud 91405 Orsay Cedex France Fax 33 1 69156777 Tel 33 1 69157909 E-mail lwlindqvist@ Present address Physics Department Faculty of Sciences and Arts Targoviste Romania Received 10 October 2009 revised 2 December 2009 accepted 7 December 2009 doi The reactions of the flavin semiquinone generated by laser-induced stepwise two-photon excitation of reduced flavin have been studied previously El Hanine-Lmoumene C Lindqvist L. 1997 Photochem Photobiol 66 591-595 using time-resolved spectroscopy. In the present work we have used the same experimental procedure to study the flavin semiquinone in rat kidney long-chain hydroxy acid oxidase and in the flavodehydrogenase domain of flavocytochrome b2 FDH two homologous flavoproteins belonging to the family of FMN-dependent L-2-hydroxy acid-oxidizing enzymes. For both proteins pulsed laser irradiation at 355 nm of the reduced enzyme generated initially the neutral semiquinone which has rarely been observed previously for these enzymes and hydrated electron. The radical evolved with time to the anionic semiquinone that is known to be stabilized by these enzymes at physiological pH. The deprotonation kinetics were biphasic with durations of 1-5 is and tens of microseconds respectively. The .