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.

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 D...