Ultrafast fluorescence dynamics of FMN-binding protein from Desulfovibrio vulgaris (Miyazaki F) and its site-directed mutated proteins

Ultrafast fluorescence dynamics of FMN in FMN-binding protein (FMN-bp), and its mutated proteins, W32Y and W32A, were investigated by the fluorescence up-conversion method. Fluorescence lifetimes were 167 fs (96%) and 1.5 ps (4%) in wild-type FMN-bp (WT), and 3.4 ps (23%), 18.2 ps (74%), and 96 ps (3%) at 530 nm in W32Y, and 30.1 ps in W32A. The fluorescence lifetime of W32A, in which Trp-32 was absent, was about 140 times longer than that of WT. Tyr-32 in W32Y was not so effective quencher as Trp-32 in WT. This was explained in terms of different ionization potentials of quenchers and average donor–acceptor distances in the protein.