15N,18O tracer studies on the activation of nitrite by denitrifying bacteria. Nitrite/water-oxygen exchange and nitrosation reactions as indicators of electrophilic catalysis.

tory to reduction relative to nitrous acid (6).If nitrite reductases render enzyme-bound nitrite more like HNOz (or HzN02+ or NO'), presumably by interaction with a metal center, one might expect to observe catalysis at pH 7 of reactions otherwise subject to acid catalysis in acidic solution, namely, nitrosation (nitrosyl transfer) reactions and nitrite/ HzO-"O exchange (7-14).Reduction of nitrite via the initia1 formation of a ferrous porphyrin-nitrosyl compound has been proposed by Averill and Tiedje ( 27) on theoretical grounds.We report herein catalysis of nitrosation reactions and oxygen exchange by denitrifying bacteria. EXPERIMENTAL PROCEDURESMateriaZ~-[~'N]NaNO~ (99 atom %) was obtained from Stohler Isotope Chemical Co.(Waltham, MA) and its chemical and isotopic purities were as reported previously (15).H2'"O (97 atom %) was obtained from Merck, Sharp and Dohme.Mass spectrometric analysis of the H2180 by C. Colson of this department confirmed the supplier's statement of isotopic purity.Antimycin A and rotenone were obtained from Sigma Chemical Co. and stock solutions of 10 and 2 mg X ml-I, respectively, were prepared in 958 ethanol immediately before use.Rotenone solutions were protected from light to prevent photodecomposition ( 16).Hydroxylamine stock solutions, 1 M, containing 0.8 M free base were prepared from recrystallized hydroxylamine hydrochloride immediately before use and maintained under anaerobic conditions in order to prevent spontaneous decomposition of the free base (17).GC/MS' analysis of the gas ever such solutions confirmed that no decomposition of NH,OH occurred over a period of at least 2 h.Bacteria-Paracoccus denitrificans (formerly Micrococcus) prototype strain ATCC 19367, Pseudomonas denitrificans prototype strain ATCC 13867, Pseudomonas stutzeri strain ATCC 11607, and Pseudomonas aureofaciens strain ATCC 13985 (assigned to Pseudomonas fluorescens biotype E (18)) were used.The latter bacterium was obtained from J.