Elucidation of the Coordination Chemistry of the Enzyme-Substrate Complex of Catechol 1,2-Dioxygenase by NMR Spectroscopy

Lawrence Que, Randall B. Lauffer, John B. Lynch, Bruce P. Murch, Joseph W. Pyrz

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31 Scopus citations

Abstract

The coordination chemistry of the active site iron in catechol 1,2-dioxyygenase (CTD) from Pseudomonas putida (ATCC 23974) has been probed with NMR spectroscopy. The high-spin ferric center gives rise to paramagnetically shifted resonances in the range of 105 to -67 ppm, assigned to protons on the iron ligands. Observed endogenous ligand resonances are assigned to β-CH2protons of tyrosines and histidines. Exogenous ligand resonances are also observed, particularly those of methyl protons on substrates and inhibitors. The methyl resonance of the CTD-4-methylcatechol complex is found at 105 ppm. Comparisons of the 105-ppm shift with corresponding ones in models and in CTD-methylphenol and other CTD-substrate complexes indicate that 4-methylcatechol coordinates to the iron via 01 alone and that both catecholate protons are likely to be dissociated. The implications of the observations are discussed in terms of the proposed substrate activation mechanism.

Original languageEnglish (US)
Pages (from-to)5381-5385
Number of pages5
JournalJournal of the American Chemical Society
Volume109
Issue number18
DOIs
StatePublished - Sep 1 1987

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