title: Distribution of valence electrons of the flavin cofactor in NADH-cytochrome b5reductase.
abstract: Flavin compounds such as flavin adenine dinucleotide (FAD), flavin mononucleotide and riboflavin make up the active centers in flavoproteins that facilitate various oxidoreductive processes. The fine structural features of the hydrogens and valence electrons of the flavin molecules in the protein environment are critical to the functions of the flavoproteins. However, information on these features cannot be obtained from conventional protein X-ray analyses at ordinary resolution. Here we report the charge density analysis of a flavoenzyme, NADH-cytochrome b5reductase (b5R), at an ultra-high resolution of 0.78 Å. Valence electrons on the FAD cofactor as well as the peptide portion, which are clearly visualized even after the conventional refinement, are analyzed by the multipolar atomic model refinement. The topological analysis for the determined electron density reveals the valence electronic structure of the isoalloxazine ring of FAD and hydrogen-bonding interactions with the protein environment. The tetrahedral electronic distribution around the N5 atom of FAD in b5R is stabilized by hydrogen bonding with CαH of Tyr65 and amide-H of Thr66. The hydrogen bonding network leads to His49 composing the cytochrome b5-binding site via non-classical hydrogen bonds between N5 of FAD and CαH of Tyr65 and O of Tyr65 and CβH of His49.
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Kiyofumi
authors.initials: K
authors.lastname: Takaba
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Takeda
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Kosugi
authors.affiliation: Quantum Beam Science Research Directorate, National Institutes for Quantum and Radiological Science and Technology, Tokai-mura, Ibaraki 319-1106, Japan.
authors.forename: Taro
authors.initials: T
authors.lastname: Tamada
authors.affiliation: Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Kunio
authors.initials: K
authors.lastname: Miki
mesh_terms: D000818:Animals; D042966:Cytochrome-B(5) Reductase / Q000737:chemistry*; D004138:Dinitrocresols / Q000737:chemistry*; D004583:Electrons; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006860:Hydrogen Bonding; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013552:Swine
chemical_list: D004138:Dinitrocresols; D011994:Recombinant Proteins; C024132:4,6-dinitro-o-cresol; D042966:Cytochrome-B(5) Reductase