Structural Flexibility and Electronic Tuning in N₅-Substituted Anionic Reduced Flavins: Implications for Flavoenzyme Intermediates

N 5 -substituted reduced flavins are pivotal and yet underexplored intermediates in flavoenzyme catalysis, which hold untapped potential for advancing enzymatic and catalytic processes. Here, we combine steady-state and ultrafast transient absorption spectroscopy with DFT/TDDFT simulations to explore the structural and electronic dynamics of several nonfluorescent anionic reduced flavin derivatives involved in flavoenzyme processes. Unlike the broad, featureless absorption spectrum of FADH –, N 5 -substituted flavins exhibit distinct blue-shifted absorption bands at 330 or 370 nm, reflecting substantial electronic reorganization. Our theoretical results challenge the conventional view that isoalloxazine ring bending along the N 5 –N 10 axis governs the absorption properties of the molecule, and demonstrate instead that HOMO orbital stabilization is the critical factor. Ultrafast spectroscopy reveals transient absorption bands above 450 nm, corresponding to different isoalloxazine bend conformations, which converge into a single band at ∼500 nm within tens of picoseconds. This evolution is assigned to a rapid structural relaxation toward a more planar configuration. Along this path the system relaxes to the ground state through a conical intersection (CI) whose position is modulated by the nature of the N 5 substituent. These findings fundamentally reshape our understanding of flavin photophysics.