No abstract

The structural basis for the temperature-induced transition in the D-amino acid oxidase (DAAO) monomer from pig kidney was studied by means of molecular dynamic simulations (MDS). The center to center (Rc) distances between the isoalloxazine ring (Iso) and all aromatic amino acids (Trp and Tyr) were calculated at 10 °C and 30 °C. Rc was shortest in Tyr224 (0.82 and 0.88 nm at 10 and 30 °C, respectively), and then in Tyr228. Hydrogen bonding (H-bond) formed between the Iso N1 and Gly315 N (peptide), between the Iso N3H and Leu51 O (peptide) and between the Iso N5 and Ala49 N (peptide) at 10 °C, whilst no H-bond was formed at the Iso N1 and Iso N3H at 30 °C. The H-bond of Iso O4 with Leu51 N (peptide) at 10 °C switched to that with Ala49 N (peptide) at 30 °C. The reported fluorescence lifetimes (228 and 182 ps at 10 and 30 °C, respectively) of DAAO were analyzed with Kakitani and Mataga (KM) ET theory. The calculated fluorescence lifetimes displayed an excellent agreement with the observed lifetimes. The ET rate was fastest from Tyr224 to the excited Iso (Iso*) at 10 °C and from Tyr314 at 30 °C, despite the fact that the Rc was shortest between Iso and Tyr224 at both temperatures. This was explained by the electrostatic energy in the protein. The differences in the observed fluorescence lifetimes at 10 and 30 °C were ascribed to the differences in electron affinity of the Iso* at both temperatures, in which the free energies of the electron affinity of Iso* at 10 and 30 °C were -8.69 eV and -8.51 eV respectively. The other physical quantities related to ET did not differ appreciably at both temperatures. The electron affinities at both temperatures were calculated with a semi-empirical molecular orbital method (MO) of PM6. Mean calculated electron affinities over 100 snapshots with 0.1 ps intervals were -7.69 eV at 10 °C and -7.59 eV at 30 °C. The difference in the calculated electron affinities, -0.11 eV, was close to the observed difference in the free energies, -0.18 eV. The present quantitative analysis predicts that the highest ET rate can occur from a donor with longer donor-acceptor distance, which was explained by differences in electrostatic energy.

No abstract

Pyranose 2-oxidase (P2O) from Trametes multicolor contains FAD as cofactor, and forms a tetramer. The protein structure of a mutated P2O, T169S (Thr169 is replaced by Ser), in solution was studied by means of molecular dynamics simulation and analyses of photoinduced electron transfer (ET) from Trp168 to excited isoalloxazine (Iso*), and was compared with wild type (WT) P2O. Hydrogen bonding between Iso and nearby amino acids was very similar as between T169S and WT protein. Distances between Iso and Tyr456 were extremely heterogeneous among the subunits, 1.7 (1.5 in WT) in subunit A (Sub A), 0.97 (2.2 in WT) in Sub B, 1.3 (2.1 in WT) in Sub C, 1.3 nm (2.0 in WT) in Sub D. Mean values of root of mean square fluctuation over all residues were greater by four times than those in WT. This suggests that the protein structure of T169S is much more flexible than that of WT. Electrostatic (ES) energies between Iso anion in one subunit and ionic groups in the entire protein were evaluated. It was found that more than 50% of the total ES energy in each subunit is contributed from other subunits. Reported fluorescence decays were analyzed by a method as WT, previously reported. Electron affinities of Iso* in T169S were appreciably higher than those in WT. Static dielectric constants near Iso and Trp168 were also quite higher in T169S than those in WT.

The structural difference between two subunits of D-amino acid oxidase dimer from porcine kidney was studied by molecular dynamics simulation (MDS) and rate of photoinduced electron transfer (ET) from aromatic amino acids as tyrosines (Tyr) and tryptophanes (Trp) to the excited isoalloxazine (Iso*). The donor-acceptor distances (Rc) between isoalloxazine (Iso) and the donors were shortest in Tyr224 (0.74 nm) in Sub A at 10 °C (Sub A10), in Tyr224 (0.79 nm) in Sub B at 10 °C (Sub B10), in Tyr228 (0.85 nm) in Sub A at 30 °C (Sub A30), and in Tyr224 (0.72 nm) in Sub B at 30 °C (Sub B30). The Rcs were mostly shorter in the dimer than those in the monomer. Hydrogen bonding (H-bond) pairs between Iso and surrounding amino acids varied with the subunit and temperature. O2 of the Iso ring formed an H-bond exclusively with Thr317OG1 (side chain) in both Sub A10 and Sub A30, while it formed with Gly315N (peptide), Leu316N and Thr317N in Sub B10 and Sub B30. N3H of Iso formed an H-bond with Leu51O (peptide) in Sub A10 and Sub A30, but not in Sub B10 and Sub B30. Electron affinity of Iso* was appreciably lower in Sub A10 compared to Sub B10, while it was opposite at 30 °C. ET rate to Iso* was fastest from Tyr224 in Sub A10, while it was fastest from Tyr314 in Sub B10. The ET rate was fastest from Tyr314 in Sub A30, while it was fastest from Tyr224 in Sub B30. The greater ET rates in the dimer as compared to those in the monomer were elucidated with shorter Rc in the dimer as compared to the monomer. The static dielectric constants inside the subunits and the static dielectric constant between Iso and Tyr224 or Tyr228 were not different appreciably. A few water molecules and sometimes an amino acid were located between Iso and Tyr224, which may be the reason why the dielectric constant of the entire subunits did not differ from that between Iso and Tyr224.