Reduced Flavin in Aqueous Solution Is Nonfluorescent.

Flavins are blue-light-absorbing chromophores with rich redox activity. Biologically, the most important are riboflavin (vitamin B2), flavin mononucleotide, and flavin adenine dinucleotide, the latter two of which are catalytic cofactors in enzymes. Flavins pivot between oxidized, one electron-, and two electron-reduced forms in different protonation states, depending on enzymatic requirements. Some flavoenzymes use light as a reagent for chemical bond formation, photoinduced electron transfer, or conformational changes required for light-sensitive signaling. Therefore, the photochemistry and photophysics of flavins have received wide attention. Fluorescence from oxidized flavin is often used to detect and track changes in flavin oxidation states. However, there have been conflicting reports over the past 45 years as to whether reduced flavin in solution has detectable fluorescence. Here, using single photon counting emission spectroscopy with rigorous sample preparation, we show definitively that reduced flavins are essentially nonfluorescent, having a quantum yield more than three orders of magnitude lower than oxidized flavin. This result will force a re-evaluation of experiments and models that assumed otherwise.

Comparing ultrafast excited state quenching of flavin 1,N6-ethenoadenine dinucleotide and flavin adenine dinucleotide by optical spectroscopy and DFT calculations.

Flavins are photoenzymatic cofactors often exploiting the absorption of light to energize photoinduced redox chemistry in a variety of contexts. Both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are used for this function. The study of these photoenzymes has been facilitated using flavin analogs. Most of these analogs involve modification of the flavin ring, and there is recent evidence that adenine (Ade)-modified FAD can affect enzyme turnover, but so far this has only been shown for enzymes where the adenine and flavin rings are close to each other in a stacked conformation. FAD is also stacked in aqueous solution, and its photodynamics are quite different from unstacked FAD or FMN. Oxidized photoexcited FAD decays rapidly, presumably through PET with Ade as donor and Fl* as acceptor. Definitive identification of the spectral signatures of Ade∙+ and Fl∙- radicals is elusive. Here we use the FAD analog Flavin 1,N6-Ethenoadenine Dinucleotide (εFAD) to study how different photochemical outcomes depend on the identity of the Ade moiety in stacked FAD and its analog εFAD. We have used UV-Vis transient absorption spectroscopy complemented by TD-DFT calculations to investigate the excited state evolution of the flavins. In FAD*, no radicals were observed, suggesting that FAD* does not undergo PET. εFAD* kinetics showed a broad absorption band that suggests a charge transfer state exists upon photoexcitation with evidence for radical pair formation. Surprisingly, significant triplet flavin was produced from εFAD* We hypothesize that the dipolar (ε)Ade moieties differentially modulate the singlet-triplet energy gap, resulting in different intersystem crossing rates. The additional electron density on the etheno group of εFAD supplies better orbital overlap with the flavin S1 state, accelerating charge transfer in that molecule.

Choosing Fluorescent Probes and Labeling Systems.

Fluorescence microscopy is advantageous for investigating biological processes and mechanisms in living cells. One of the most important considerations when designing an experiment is the selection of an appropriate fluorescent probe. Equally important is deciding how the probe will be attached to the protein of interest. The advantages and disadvantages of different fluorescent probe types and their respective labeling methods are discussed to provide an overview on selecting appropriate fluorophores and labeling systems for fluorescence-based assays. Protocols are outlined when appropriate.