Salicylate is an important intermediate in the bacterial degradation of polycyclic aromatic hydrocarbons and salicylate hydroxylases play essential roles in linking the peripheral and ring-cleavage catabolic pathways. Unlike the well-characterized salicylate 1-hydroxylases, the rarely occurred salicylate 5-hydroxylase (S5H) has not been characterized in detail. In this study, the three-component Fe-S protein complex (NagAaGHAb) of S5H from Ralstonia sp. strain U2 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component NagGH exhibited an alpha(3)beta(3) heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per alpha subunit. NagAa is the ferredoxin-NADP(+) reductase component containing flavin and plant type [2Fe-2S] cluster. The ferredoxin component NagAb was characterized as a [2Fe-2S] dimer which remains remarkably stable in denaturing gel electrophoresis after being heated at 100 A degrees C for 1 h. Purified NagAa and NagAb, NagGH catalyzed the hydroxylation of salicylate to gentisate with a specific activity of 107.12 A +/- 14.38 U/g and showed an apparent K (m) for salicylate of 102.79 A +/- 27.20 mu M and a similar K (m) value for both NADH and NADPH (59.76 A +/- 7.81 mu M versus 56.41 A +/- 12.76 mu M). The hydroxylase exhibited different affinities for two hydroxysalicylates (2,4-dihydroxybenzoate K (m) of 93.54 A +/- 18.50 mu M versus 2,6-dihydroxybenzoate K (m) of 939.80 A +/- 199.46 mu M). Interestingly, this S5H also showed catalytic activity to the pollutant 2-nitrophenol and exhibited steady-state kinetic data of the same order of magnitude as those for salicylate. This study will allow further comparative studies of structure-function relationships of the ring hydroxylating mono- and di-oxygenase systems.