Identification of sulfur as component of the EPR signal at g = 1.94 by isotopic substitution

Abstract

Catechol 2,3-dioxygenase (XylE) is a component of the TOL plasmid-encoded pathway for the degradation of toluene and xylenes and catalyzes the dioxygenolytic cleavage of the aromatic ring. Purified XylE is oxygen-sensitive and unstable in vitro, particularly in the presence of substituted catechol substrates, but it is stabilized in vivo by another protein, XylT, encoded by the xylT gene located just upstream of xylE. In this study, we have purified to homogeneity the XylT product from a recombinant Escherichia coli strain containing a hyperexpressible xylT gene and characterized it as a novel [2Fe-2S] ferredoxin. It is the first example of a soluble ferredoxin with a net positive charge at neutral pH. The EPR signal of the iron sulfur cluster has rhombic symmetry as is the case for plant-type ferredoxins, but the XylT absorbance spectrum resembles more closely that of adrenodoxin. The midpoint redox potential was determined to be −373 ± 6 mV, at pH 8.5. XylT was unusually unstable for a [2Fe-2S] ferredoxin, with half-lives of 69 min at 25 °C in air and 70 min at 37 °C in argon. With photochemically reduced 5-deazaflavin for the controlled generation of reductant, it was demonstrated that XylT mediates the rapid reactivation of purified inactive catechol 2,3-dioxygenase in vitro. Inactivation of XylE by 4-methylcatechol resulted in oxidation of the active site iron to a high spin ferric state that was detectable by EPR. Spectroscopic evidence presented here demonstrates that XylT reactivates XylE through reduction of the iron atom in the active site of the enzyme. It is the first instance of a ferredoxin-mediated reactivation of an enzyme. The level of expression of XylT in Pseudomonas putida mt2 cells is low and the calculated XylT/XylE molar ratio is consistent with the proposal that XylE reactivation involves catalytic nonstoichiometric amounts of XylT.