EPR Studies of Chlorocatechol 1,2-Dioxygenase: Evidences of Iron Reduction during Catalysis and of the Binding of Amphipatic Molecules

Abstract

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.

Ferric species equilibrium of the giant extracellular hemoglobin of Glossoscolex paulistus in alkaline medium: HALS hemichrome as a precursor of pentacoordinate species

“…This evidence is in agreement with the UV-vis results, indicating a representative presence in equilibrium of a pentacoordinate ferric species. Furthermore, a very intense signal around g = 4.3 is observed in the higher pH values, which has been identified in other ferric proteins and it has been assigned to non-heme iron centers [56,57].…”

Stabilization and functionalization of iron oxide nanoparticles for biomedical applications

“…87 Because of the biological relevance of DOPA/Fe 3+ and dopamine/Fe 3+ complexes, their structure [88][89][90] and electronic interactions 91,92 have been studied in detail. Furthermore, the crystallographic and electronic structure of peptides, 93 proteins 94 and models for catechol dioxygenases complexed with iron ions 89,[95][96][97] have been reported. Despite their popularity as anchors fueled by biological inspiration and initial investigations on their application as dispersant anchors for iron oxide NPs, the suitability of catechols as anchors to stabilize iron oxide NPs is debated.…”

Estudos da correlação estrutura-função da enzima Clorocatecol 1,2-Dioxigenase de Pseudomonas putida

“…[13][14][15][16][17] Neste sítio foi detectada uma densidade eletrônica compatível com a estrutura de um ácido graxo e não à de um fosfolipídio como relatado na literatura [13][14][15][16] . Além disso, trabalhos realizados anteriormente em nosso grupo de pesquisa mostraram que a possível delipidação da proteína, acarretaria uma perda da atividade específica, bem como levaria a uma menor afinidade da enzima pelo seu substrato 19 .…”

“…Esta hipótese fora levantada pela inesperada presença de moléculas anfipáticas na interface do dímero que constitui a estrutura da Pp 1,2-CCD. (19) Ou seja, se há uma molécula com tais características na estrutura da enzima, interessa-nos saber o seu papel sobre a catálise enzimática (abordado nas seções anteriores), mas também suscita a indagação acerca de um provável mecanismo regulatório que envolvesse a interação com membranas biológicas. Sendo assim, escolhemos o sistema mimético de membranas mais simples com o qual se pode trabalhar, qual seja micelas de lisofosfolipídios.…”