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

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 LD = 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 pro‐tolytic 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 de‐protonation of this radical. The value pK a = 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.