Excited-State Properties of Fully Reduced Flavins in Ferredoxin-NADP Oxidoreductase.

The fully reduced flavin cofactor (FAD) in ferredoxin-NADP oxidoreductase (FNR) is a functional intermediate that displays different catalytic and steady-state spectral properties for enzymes from (FNR), (FNR), and (FNR). Using ultrafast spectroscopy, we reveal that at physiological pH, photoexcited FAD in FNR and FNR exhibits unprecedentedly fast decays (dominantly in 6 and 8 ps, respectively), whereas in FNR the decay is much slower (∼400 ps), as in other flavoproteins. Correlating these observations with the protonation states of FAD and the dynamic properties of the protein environment, we conclude that the excited state of neutral FAD can be intrinsically short-lived even in proteins, contrasting with the well-documented behavior of the anionic form that systematically displays markedly increased excited-state lifetime upon binding to proteins. This work provides new insight into the photochemistry of fully reduced flavins, which are emerging as functional initial states in bioengineered photocatalysts.