Stepwise Two-Photon Excitation of 1,5-Dihydroflavin Mononucleotide: Study of Flavosemiquinone Properties

Abstract

Photoionization of 1,5-dihydroflavin mononucleotide in aqueous solution, leading to formation of hydrated electrons and flavosemiquinone radicals, was studied by nanosecond laser photolysis (excitation at λ = 354.7 nm). Analysis of the laser fluence dependence of the photoionization yield indicates that the reaction is a stepwise two-photon reaction with the fluorescent state of the flavin as intermediate step. Extinction coefficients of the neutral and anionic flavosemiquinone were obtained from transient absorbance measurements at 310-660 nm. The protolytic dissociation constant of the neutral radical was determined from a study at pH 7.3-10.3 of the spectral evolution in the submicrosecond time range due to deprotonation of this radical. The value pKa = 8.7 ± 0.1 was obtained. The formation of tautomeric species of the neutral flavosemiquinone was suggested based on the existence of a spectral evolution of this species during the first microseconds after excitation.