Prediction of Isotope Effects for Anticipated Intermediate Structures in the Course of Bacterial Denitrification

Abstract

P re d ic tio n o f Iso to p e E ffe c ts fo r A n tic ip a te d I n te r m e d ia te S tr u c tu r e s in th e C o u rs e o f B a c te ria l D e n itrific a tio n M. A. Morgenstern and R. L. Schowen Department of Chemistry, University of Kansas, Lawrence, Kansas 66045-0046, USA Z. Naturforsch. 44a, 450 458 (1989); received January 10, 1989 This paper is dedicated to Professor Jacob Bigeleisen on the occasion of his 70th birthday Vibrational-analysis methods have been used to estimate the equilibrium 14N/15N isotope effects to be expected for conversion of nitrite anion to thirteen possible intermediate-state and productstate structures [HONO, NO + , NO, NO", FeNO, 0N*N02, 0*NN02, 0 2NN02, ONO*N, 0*N0N, ONNO, *NNO, N*NO] in the reduction of nitrite ion to nitrous oxide denitrifying bacteria. The results, taken in combination with previous experimental isotope-effect and tracer studies of the Pseudomonas stutzeri and related systems, are consistent with a suggestion that a second nitrite anion enters the enzyme-catalytic cycle at the stage of a nitrosyl-ion intermediate but re-emerges after entry of the reducing electrons; the product nitrous oxide is then formed by disproportionation of enzymically generated hyponitrous acid. The calculations are consistent with contributions, under different experimental conditions, of several different transition states to limit ing the rate of the enzymic reaction. These transition states (and the corresponding experimental conditions) are the transition states for N -O fission in the generation of a mononitrogen electrophilic species from nitrite anion (high reductant, high nitrite concentrations), for attack of nitrite on this electrophile (high reductant, low nitrite concentrations) and for electron transfer to a dinitrogen-trioxide-like species (low reductant concentration).

Is the isotopic composition of nitrous oxide an indicator for its origin from nitrification or denitrification? A theoretical approach from referred data and microbiological and enzyme kinetic aspects

“…However, in our opinion, this interpretation is erroneous because the decay of the isotopomers of a symmetrical intermediate of the NO reduction must be accompanied by intramolecular isotope effects and cause the simultaneous enrichment of 18 O and of 15 N in the central position of N 2 O, thus leading to an enrichment of the heavy isotope of both elements in the formed N 2 O. Isotopic shifts in the infrared spectra17, 18 of isotopomers of − ONNO − prove distinct differences of the binding constants between the isotopes, prerequisites for isotope effects on the binding fissions involved. Their existence is confirmed by calculations by Morgenstern and Schowen19 for the overall nitrogen equilibrium isotope effects of the formation of 15 N 14 NO and 14 N 15 NO, respectively, from NO; the calculated values were 1.0050 and 0.9601, respectively. Hence H 16 O 14 N 14 N 18 OH will preferably lead to 14 N 14 N 18 O+H 16 OH, and H 16 O 14 N 15 N 16 OH will preferably yield 14 N 15 N 16 O+H 16 OH.…”

“…NO can exchange O atoms with water, preferably under influence of heme‐containing nitrite reductases,21 and one can expect that the reductions of NO and of NO certainly prefer the elimination of 16 O (intramolecular isotope effect). Generally, all these reactions must hence be accompanied by isotope discriminations, mainly on the basis of kinetic isotope effects 19, 22. Recent experiments by Casciotti et al 23.…”