Conformational heterogeneity in pyranose 2-oxidase from Trametes multicolor revealed by ultrafast fluorescence dynamics

Ultrafast fluorescence dynamics of flavin adenine dinucleotide (FAD) in wild type pyranose 2-oxidase (P2O) has been investigated in solution by means of fluorescence up-conversion method. Fluorescence decays were well described by two-exponential model function. Fluorescence lifetimes were τ1 ∼ 110 fs and τ2 ∼ 360 ps, respectively. The (τ2/τ1) ratio (∼3200) was extraordinary high compared to other flavoproteins without subunit structure. The heterogeneous distribution of emission lifetimes were elucidated in terms of two different conformers of P2O; conformer 1 with τ1 and conformer 2 with τ2. Emission peaks of conformer 1 and conformer 2 were determined to be at ∼540 nm and 510 nm, respectively, using transient spectral reconstruction procedure. Using dynamics analysis by Kakitani and Mataga (KM) theory, both quenching processes were ascribed to photoinduced electron transfer (ET) reactions mainly from Trp168 to the excited isoalloxazine (Iso*) in different protein tetramers having different static dielectric constants (ɛ1 ∼ 3.25 for conformer 1 and ɛ2 ∼ 5.93 for conformer 2). The quaternary structure seems to be responsible for the observed conformational heterogeneity.