title: An extended N-H bond, driven by a conserved second-order interaction, orients the flavin N5 orbital in cholesterol oxidase.
abstract: The protein microenvironment surrounding the flavin cofactor in flavoenzymes is key to the efficiency and diversity of reactions catalysed by this class of enzymes. X-ray diffraction structures of oxidoreductase flavoenzymes have revealed recurrent features which facilitate catalysis, such as a hydrogen bond between a main chain nitrogen atom and the flavin redox center (N5). A neutron diffraction study of cholesterol oxidase has revealed an unusual elongated main chain nitrogen to hydrogen bond distance positioning the hydrogen atom towards the flavin N5 reactive center. Investigation of the structural features which could cause such an unusual occurrence revealed a positively charged lysine side chain, conserved in other flavin mediated oxidoreductases, in a second shell away from the FAD cofactor acting to polarize the peptide bond through interaction with the carbonyl oxygen atom. Double-hybrid density functional theory calculations confirm that this electrostatic arrangement affects the N-H bond length in the region of the flavin reactive center. We propose a novel second-order partial-charge interaction network which enables the correct orientation of the hydride receiving orbital of N5. The implications of these observations for flavin mediated redox chemistry are discussed.
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia, 6009 Australia.
authors.forename: Emily
authors.initials: E
authors.lastname: Golden
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia, 6009 Australia.
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Yu
authors.affiliation: Neutron Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, TN, 37831, United States of America.|Structural and Molecular Biochemistry, North Carolina State University, Raleigh, NC 27695, United States of America.
authors.forename: Flora
authors.initials: F
authors.lastname: Meilleur
authors.affiliation: Institut Laue-Langevin, 71 Avenue des Martyrs, Grenoble, 38000, France.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Blakeley
authors.affiliation: Bragg Institute, Australian Nuclear Science and Technology Organisation, Lucas Heights NSW, 2234, Australia.
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Duff
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia, 6009 Australia.
authors.forename: Amir
authors.initials: A
authors.lastname: Karton
authors.affiliation: School of Chemistry and Biochemistry, University of Western Australia, Crawley, Western Australia, 6009 Australia.
authors.forename: Alice
authors.initials: A
authors.lastname: Vrielink
mesh_terms: D000577:Amides / Q000737:chemistry; D001665:Binding Sites; D055162:Biocatalysis; D002789:Cholesterol Oxidase / Q000378:metabolism*; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000737:chemistry* / Q000378:metabolism*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D010084:Oxidation-Reduction; D013379:Substrate Specificity
chemical_list: D000577:Amides; D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; D002789:Cholesterol Oxidase