EPR, Electron Spin Echo Envelope Modulation, and Electron Nuclear Double Resonance Studies of the 2Fe2S Centers of the 2-Halobenzoate 1,2-Dioxygenase from Burkholderia (Pseudomonas) cepacia 2CBS

Abstract

The 2-halobenzoate 1,2-dioxygenase from Burkholderia (Pseudomonas) cepacia 2CBS (Fetzner, S., Müller, R., and Lingens, F.(1992) J. Bacteriol. 174, 279-290) contains both a ferredoxin-type and a Rieske-type 2Fe2S center. These two significantly different 2Fe2S clusters were characterized with respect to their EPR spectra, electrochemical properties (Rieske-type cluster with gz= 2.025, gy= 1.91, gx= 1.79, gav= 1.91, Em= −125 ± 10 mV; ferredoxin-type center with gz= 2.05, gy= 1.96, gx= 1.89, gav= 1.97, Em= −200 ± 10 mV) and pH dependence thereof. X band electron spin echo envelope modulation and electron nuclear double resonance spectroscopy was applied to study the interaction of the Rieske-type center of the 2-halobenzoate 1,2-dioxygenase with 14N and 1H nuclei in the vicinity of the 2Fe2S cluster. The results are compared to those obtained on the Rieske protein of the cytochrome b6f complex (Em= +320 mV) and the water-soluble ferredoxin (Em= −430 mV) of spinach chloroplasts, as typical representatives of the gav=1.91 and gav=1.96 class of 2Fe2S centers. Properties common to all Rieske-type clusters and those restricted to the respective centers in bacterial oxygenases are discussed. The 2-halobenzoate 1,2-dioxygenase from Burkholderia (Pseudomonas) cepacia 2CBS (Fetzner, S., Müller, R., and Lingens, F.(1992) J. Bacteriol. 174, 279-290) contains both a ferredoxin-type and a Rieske-type 2Fe2S center. These two significantly different 2Fe2S clusters were characterized with respect to their EPR spectra, electrochemical properties (Rieske-type cluster with gz= 2.025, gy= 1.91, gx= 1.79, gav= 1.91, Em= −125 ± 10 mV; ferredoxin-type center with gz= 2.05, gy= 1.96, gx= 1.89, gav= 1.97, Em= −200 ± 10 mV) and pH dependence thereof. X band electron spin echo envelope modulation and electron nuclear double resonance spectroscopy was applied to study the interaction of the Rieske-type center of the 2-halobenzoate 1,2-dioxygenase with 14N and 1H nuclei in the vicinity of the 2Fe2S cluster. The results are compared to those obtained on the Rieske protein of the cytochrome b6f complex (Em= +320 mV) and the water-soluble ferredoxin (Em= −430 mV) of spinach chloroplasts, as typical representatives of the gav=1.91 and gav=1.96 class of 2Fe2S centers. Properties common to all Rieske-type clusters and those restricted to the respective centers in bacterial oxygenases are discussed.