An upper limit to the fluorescence quantum yield for reduced flavins – a spectroscopic approach to better understand photoflavoproteins

Abstract

Flavins are blue‐light absorbing chromophores with rich redox activity, found in oxidized, one electron‐ and two electron‐reduced forms. Some, namely flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are used by enzymes as catalytic cofactors. Conflicting literature regarding reduced flavin fluorescence emission includes a plethora of results, from some finding there is negligible fluorescence to others observing significantly blue‐shifted emission. This discrepancy is of consequence because mechanistic kinetic models of photoflavoproteins derived from optical spectroscopy rely on a proper assignment of this emission. Reduced flavins are common redox cofactors in cells, and any fluorescence‐based imaging of this molecule in vivo requires an accurate estimate of its emission quantum yield and spectrum. Here, the photo‐induced reduction of HPLC‐purified FMN and other relevant flavins, in aqueous solutions (at pH 5.0 and 8.5) under aerobic and anaerobic conditions, has been studied via steady‐state absorption and fluorescence measurements. An exogenous reducing agent, ethylenediaminetetraacetic acid (EDTA), was used to photo‐reduce the flavins under investigation. Facile and complete reduction of FMN (~105‐fold) is achieved under anaerobic conditions and insignificant amounts of reduced fluorescence emission is observed, allowing for calculation of the upper limit of the quantum yield of reduced flavin. The low quantum yield (~10‐4) obtained is an important finding when considering flavoproteins, especially when studying them spectroscopically.