Spectroscopic and Electronic Structure Studies of 2,3-Dihydroxybiphenyl 1,2-Dioxygenase:  O2 Reactivity of the Non-Heme Ferrous Site in Extradiol Dioxygenases

• Published in: Journal of the American Chemical Society 2003-09, Vol.125 (37), p.11214-11227
• Main Authors: Davis, Mindy I, Wasinger, Erik C, Decker, Andrea, Pau, Monita Y. M, Vaillancourt, Frédéric H, Bolin, Jeffrey T, Eltis, Lindsay D, Hedman, Britt, Hodgson, Keith O, Solomon, Edward I
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Binding Sites; Biological and medical sciences; Dioxygenases - chemistry; Dioxygenases - metabolism; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Iron - metabolism; Models, Molecular; Oxidoreductases; Oxygen - metabolism; Protein Conformation; Pseudomonas - enzymology; Spectrum Analysis; Substrate Specificity
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja029746i

The extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHBD, EC 1.13.11.39), has been studied using magnetic circular dichroism (MCD), variable-temperature variable-field (VTVH) MCD, X-ray absorption (XAS) pre-edge, and extended X-ray absorption fine structure (EXAFS) spectroscopies, which are analogous to methods used in earlier studies on the extradiol dioxygenase catechol 2,3-dioxygenase [Mabrouk et al. J. Am. Chem Soc. 1991, 113, 4053−4061]. For DHBD, the spectroscopic data can be correlated to the results of crystallography and with the results from density functional calculations to obtain detailed geometric and electronic structure descriptions of the resting and substrate (DHB) bound forms of the enzyme. The geometry of the active site of the resting enzyme, square pyramidal with a strong Fe−glutamate bond in the equatorial plane, localizes the redox active orbital in an orientation appropriate for O2 binding. However, the O2 reaction is not favorable, as it would produce a ferric superoxide intermediate with a weak Fe−O bond. Substrate binding leads to a new square pyramidal structure with the strong Fe−glutamate bond in the axial direction as indicated by a decrease in the 5Eg and increase in the 5T2g splitting. Electronic structure calculations provide insight into the relative lack of dioxygen reactivity for the resting enzyme and its activation upon substrate binding.