EPR studies of chlorocatechol 1,2-dioxygenase: evidences of iron reduction during catalysis and of the binding of amphipatic molecules.
Chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp 1,2-CCD) is a dioxygenase responsible for ring cleavage during the degradation of recalcitrant aromatic compounds. We determined the zero-field splitting of the Fe(III) cofactor (|D| = 1.3 +/- 0.2 cm(-1)) by electron paramagnetic resonance (EPR) experiments that along with other structural data allowed us to infer the Fe(III) coordination environment. The EPR spectrum of the ion shows a significantly decrease of the g = 4.3 resonance upon substrate binding. This result is rationalized in terms of a mechanism previously proposed, where catechol substrate is activated by Fe(III), yielding an exchange-coupled Fe(II)-semiquinone (pair). The Pp 1,2-CCD capacity of binding amphipatic molecules and the effects of such binding on protein activity are also investigated. EPR spectra of spin labels show a protein-bound component, which was characterized by means of spectral simulations. Our results indicate that Pp 1,2-CCD is able to bind amphipatic molecules in a channel with the headgroup pointing outwards into the solvent, whereas the carbon chain is held inside the tunnel. Protein assays show that the enzyme activity is significantly lowered in the presence of stearic-acid molecules. The role of the binding of those molecules as an enzyme activity modulator is discussed.
Citadini
Ana P S
AP
Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, São Carlos, SP, Brazil.
Pinto
Andressa P A
AP
Araújo
Ana P U
AP
Nascimento
Otaciro R
OR
Costa-Filho
Antonio J
AJ
D001665:Binding Sites; D001703:Biophysics / Q000379:methods*; D002244:Carbon / Q000737:chemistry; D002384:Catalysis; D002396:Catechols / Q000737:chemistry; D049308:Dioxygenases / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D007501:Iron / Q000737:chemistry*; D008280:Magnetics; D010105:Oxygenases / Q000737:chemistry; D011485:Protein Binding; D016958:Pseudomonas putida / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature
D002396:Catechols; D011994:Recombinant Proteins; D002244:Carbon; D007501:Iron; D010105:Oxygenases; D049308:Dioxygenases; C044173:chlorocatechol 1,2-dioxygenase; C034221:catechol
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