Dynamics of Flavin in Flavocytochrome b2: A Fluorescence Study

Abstract

Abstract— The dynamics of the flavin bound to the flavocytochrome b2 from Hansenula anomala were studied by fluorescence intensity quenching and quenching emission anisotropy with iodide. The fluorescence intensity of bound flavin is decreased 13‐fold as compared to the free molecule. The remaining fluorescence decays with two lifetimes equal to 0.963 ± 0.040 and 4.635 ± 0.008 ns and fractional intensities of 0.036 ± 0.002 and 0.964 ± 0.002, respectively. The bimolecular diffusion constant was found to be 3.33 × 109M‐1 s‐1 when the flavin is bound to the enzyme and 8.3 × 109Mv s‐1 when the flavin is free in solution. Thus, the flavin in flavocytochrome b2 is accessible to the solvent, but the amino acid residues of the binding site inhibit the diffusion of iodide. The rotational correlation time of bound flavin was found to be 2.015 ± 0.365 ns, a value higher than that (155 ps) of free flavin in solution. Our results are discussed on the basis of local dynamics of the flavin.