Spectroscopic characterization of substrate binding to the oxygenase component of benzoate 1,2-dioxygenase

M. D. Wolfe, D. J. Altier, J. D. Lipscomb

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Benzoate 1.2-dioxygenase (BZD) catalyzes the 2 e- reduction, cleavage and insertion of 02 into benzoate to form the cis-diol cyclohexadiene product. BZD consists of two components: a monomeric oxidoreductase (BZDR) and an (0.'3)3 oxygenase (BZDO). BZDO has one Rieske-type [2Fe2S} cluster and one mononuclear, non-heine iron atom per (c,/3). We have used optical and EPR techniques to observe the binding of the 02 analog nitric oxide (NO) and benzoate to reduced BZDO (Er). UV/Vis studies of Er show new features developing at 428 and 552 nm upon addition of NO, consistent with NO coordination to ferrous ion (Er-NO). The EPR spectrum of Er-NO shows an S=3/2 species (E/D=0.013) typical of NO coordination to Fe(II). Addition of benzoate perturbs the Er-NO complex producing a new S=3/2 species (E/D=0.029). These results indicate that the mononuclear iron has at least one coordination site that can be occupied by exogenous ligands. Furthermore, the ability of benzoate to alter the environment of the Er-NO complex suggests it binds simultaneously with NO and close to the iron, consistent with 02 activation and insertion chemistry occuring at this site. ]'his research was supported by NIH grant GM-24689.

Original languageEnglish (US)
Pages (from-to)A1303
JournalFASEB Journal
Issue number9
StatePublished - Dec 1 1997


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