Characterization of three XylT-like [2Fe-2S] ferredoxins associated with catabolism of cresols or naphthalene: Evidence for their involvement in catechol dioxygenase reactivation

The xylT gene product, a component of the xylene catabolic pathway of Pseud omonas putida mt2, has been recently characterized as a novel [2Fe-2S] ferr edoxin which specifically reactivates oxygen-inactivated catechol 2,3-dioxy genase (XylE). In this study, three XylT-like proteins potentially involved in the catabolism of naphthalene (NahT) or cresols (PhhQ and DmpQ) have be en overexpressed in Escherichia coli, purified, and compared with respect t o their biochemical properties and interaction with XylE. The three XylT an alogues show general spectroscopic characteristics common to plant-type [2F e-2S] ferredoxins as well as distinctive features that appear to be typical for the XylT subgroup of these proteins. The midpoint redox potentials of the PhhQ and DmpQ proteins were -286 mV and -323 mV, respectively, Interest ingly, all purified XylT-like proteins promoted in vitro reactivation of Xy lE almost as efficiently as XylT, The interaction of XylE with XylT and its analogues was studied by cross-linking experiments using the 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide. A polypeptide band with an M-r of 46,000 , which corresponded to the cross-linked product between one XylE subunit a nd one molecule of ferredoxin, was obtained in all cases, The formation of the complex was affected by ionic strength, indicating that electrostatic f orces are involved in the dioxygenase-ferredoxin interaction, In complement ation experiments, plasmids expressing xylT or its analogues were introduce d into an XylT-null mutant of P. putida which is unable to grow on p-methyl benzoate. All transconjugants regained the wild-type phenotype, indicating that all analogues can substitute for XylT in the in vivo reactivation of X ylE. Our results provide evidence for a subgroup of [2Fe-2S] ferredoxins wi th distinct biochemical properties whose specific function is to reactivate intrinsically labile extradiol ring cleavage dioxygenases involved in the catabolism of various aromatic hydrocarbons.