PURIFICATION AND CHARACTERIZATION OF NITROBENZENE NITROREDUCTASE FROMPSEUDOMONAS PSEUDOALCALIGENES JS45

Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene as a sole sou rce of carbon, nitrogen, and energy. The catabolic pathway involves re duction to hydroxylaminobenzene followed by rearrangement to o-amino-p henol and ring fission (S. F. Nishino and J. C. Spain, Appl. Environ. Microbiol. 59:2520, 1993). A nitrobenzene-inducible, oxygen-insensitiv e nitroreductase,vas purified from extracts of JS45 by ammonium sulfat e precipitation followed by anion-exchange and gel filtration chromato graphy. A single 33-kDa polypeptide was detected by denaturing gel ele ctrophoresis. The size of the native protein was estimated to be 30 kD a by gel filtration. The enzyme is a flavoprotein with a tightly bound flavin mononucleotide cofactor in a ratio of 2 mol of flavin per mol of protein. The K-m for nitrobenzene is 5 mu M at an initial NADPH con centration of 0.5 mM. The K-m for NADPH at an initial nitrobenzene con centration of 0.1 mM is 183 mu M. Nitrosobenzene was not detected as a n intermediate of nitrobenzene reduction, but nitrosobenzene is a subs trate for the enzyme, and the specific activity for nitrosobenzene is higher than that for nitrobenzene. These results suggest that nitrosob enzene is formed but is immediately reduced to hydroxylaminobenzene. H ydroxylaminobenzene was the only product detected after incubation of the purified enzyme with nitrobenzene and NADPH. Hydroxylaminobenzene does not serve as a substrate for further reduction by this enzyme. Th e products and intermediates are consistent with two two-electron redu ctions of the parent compound. Furthermore, the low K-m and the induci ble control of enzyme synthesis suggest that nitrobenzene is the physi ological substrate for this enzyme.