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.

Crystallographic Comparison of Manganese- and Iron-Dependent Homoprotocatechuate 2,3-Dioxygenases

“…When a substrate binds, these interactions are disrupted, as also noted for the analogous residues in BPHC_LB400 (79 O . This interaction is replaced by one with the acetate group of HPCA, which forms a hydrogen bond with both H248 ND1 and R293 O .…”

Spectroscopic Studies of the Anaerobic Enzyme−Substrate Complex of Catechol 1,2-Dioxygenase

“…By contrast, difference UVRR spectroscopy has been used to show that in extradiol dioxygenases, the asymmetry in the bound catechol is induced by mono-deprotonation of the Fe(II)-bound catechol. 28 Combining difference UVRR and optical spectroscopies is likely to provide other important insights into metal ligation in solution.…”

“…A conserved second sphere histidine (His241 in 2,3-dihydroxybiphenyl 1,2-dioxygenase) residue likely assists in substrate deprotonation. 28 The origin of the asymmetry in intradiol dioxygenases is unclear. Visible resonance Raman studies of catechol 1,2-dioxygenase (C12O) showed that the spectrum of the enzyme-substrate complex is similar to the spectra of model compounds containing Fe(III)-bound dianionic catechol, suggesting that the C12O-bound catechol is also dianionic.29 Dianionic binding is consistent with the increased Lewis acidity of the ferric center.…”

Finding Intermediates in the O2 Activation Pathways of Non-Heme Iron Oxygenases

“…2,4,[6][7][8][9] This directly displaces two solvents from the "catalytic" face of the facial triad. The third solvent bond to the iron (if present) is weakened or broken by the donation of charge from the anionic catecholic ligand.…”