Ultrafast fluorescence dynamics of flavin adenine dinucleotide in pyranose 2-oxidases variants and their complexes with acetate: Conformational heterogeneity with different dielectric constants

Pyranose 2-oxidase from Trametes multicolor (P2O) catalyzes oxidations of aldopyranoses by using molecular oxygen. The enzyme is composed of four identical subunits with a native molecular weight of 270 kDa and each of which contains flavin adenine dinucleotide (FAD) as a cofactor. P2O contains the Trp168 residue near the flavin isoalloxazine ring (Iso), which can potentially act as an electron donor to the excited Iso (Iso*). Iso is covalently linked to His167 in WT P2O, but not in H167A (His167 replaced by Ala). Ultrafast fluorescence dynamics of H167A, T169S (Thr169 replaced by Ser) and acetate complexes with WT and T169S were studied by the fluorescence up-conversion method. The fluorescence decays were well described with two-exponential functions. The shorter fluorescence lifetimes (τ1) were 87–184 fs depending on an emission wavelength, and the longer lifetimes (τ2) was 120 ps without the emission wavelength-dependence in H167A. The shorter lifetimes of T169S were 92–240 fs, and the longer lifetime 15 ps. In WT P2O–acetate complex τ1 was 110 fs and τ2 50 ps, and in T169S P2O–acetate complex τ1 was 250 fs and τ2 300 ps at 530 nm. The ratio of the lifetime (τ2/τ1) was extraordinary high compared to the other flavoproteins. This result was interpreted as two conformers of P2O, Conformers 1 and 2 (Conf 1 and Conf 2), in which each was associated with τ1 and τ2, respectively. The ultra-short lifetimes were ascribed to photoinduced electron transfer (ET) mainly from Trp168 to the excited Iso (Iso*). ET rates of Confs 1 and 2 were calculated based on structural parameters reported. According to the crystal structures, distances between Iso and Trp168 were not different among the four subunits. It was found that static dielectric constants of the Conf 1 with τ1 (ɛ1 = 3.51) was different from that of the Conf 2 with τ2 (ɛ2 = 5.95). Fluorescence spectra with τ1 and τ2 were constructed based on the emission-wavelength dependent-decay functions. Emission peak with τ1 was time-dependent from the wavelengths shorter than 480 to 540 nm within 1 ps after excitation, whereas that with τ2 was steady at around 530 nm.

► Pyranose-2 oxidase forms tetramer with flavin adenine dinucleotide as a cofactor.
► Two transient fluorescence spectra were obtained with 0.1 and 50–300 ps lifetimes.
► The fluorescence lifetimes were analyzed with photoinduced electron transfer theory.
► New concept was proposed on the conformational heterogeneity.
► Two conformers exhibited different dielectric constants in five mutated isoforms.