title: Definitive evidence for monoanionic binding of 2,3-dihydroxybiphenyl to 2,3-dihydroxybiphenyl 1,2-dioxygenase from UV resonance Raman spectroscopy, UV/Vis absorption spectroscopy, and crystallography.
abstract: Ultraviolet resonance Raman spectroscopy (UVRRS), electronic absorption spectroscopy, and X-ray crystallography were used to probe the nature of the binding of 2,3-dihydroxybiphenyl (DHB) to the extradiol ring-cleavage enzyme, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHBD; EC 1.13.11.39). The lowest lying transitions in the electronic absorption spectrum of DHBD-bound DHB occurred at 299 nm, compared to 305 nm for the monoanionic DHB species in buffer. In contrast, the corresponding transitions in neutral and dianionic DHB occurred at 283 and 348 nm, respectively, indicating that DHBD-bound DHB is monoanionic. These binding-induced spectral changes, and the use of custom-designed optical fiber probes, facilitated UVRR experiments. The strongest feature of the UVRR spectrum of DHB was a Y8a-like mode around 1600 cm(-1), whose position depended strongly on the protonation state of the DHB. In the spectrum of the DHBD-bound species, this feature occurred at 1603 cm(-1), as observed in the spectrum of monoanionic DHB. Raman band shifts were observed in deuterated solvent, ruling out dianionic binding of the substrate. Thus, the electronic absorption and UVRRS data demonstrate that DHBD binds its catecholic substrate as a monoanion, definitively establishing this feature of the proposed mechanism of extradiol dioxygenases. This conclusion is supported by a crystal structure of the DHBD:DHB complex at 2.0 A resolution, which suggests that the substrate's 2-hydroxyl substituent, and not the 3-hydroxyl group, deprotonates upon binding. The structural data also show that the aromatic rings of the enzyme-bound DHB are essentially orthogonal to each other. Thus, the 6 nm blue shift of the transition for bound DHB relative to the monoanion in solution could indicate a conformational change upon binding. Catalytic roles of active site residues are proposed based on the structural data and previously proposed mechanistic schemes.
authors.affiliation: Department of Biochemistry, The University of British Columbia, 300-6174 University Boulevard, Vancouver, B.C., V6T 1Z3, Canada.
authors.forename: Frédéric H
authors.initials: FH
authors.lastname: Vaillancourt
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Barbosa
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Spiro
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Bolin
authors.forename: Michael W
authors.initials: MW
authors.lastname: Blades
authors.forename: Robin F B
authors.initials: RF
authors.lastname: Turner
authors.forename: Lindsay D
authors.initials: LD
authors.lastname: Eltis
mesh_terms: D001665:Binding Sites; D001713:Biphenyl Compounds / Q000737:chemistry / Q000378:metabolism*; D002396:Catechols / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D049308:Dioxygenases; D008958:Models, Molecular; D010105:Oxygenases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013059:Spectrum Analysis, Raman / Q000379:methods
chemical_list: D001713:Biphenyl Compounds; D002396:Catechols; C454813:2,3-dihydroxybiphenyl; D010105:Oxygenases; D049308:Dioxygenases; C074272:2,3-dihydroxybiphenyl oxygenase