Variable expression of the nitrogen isotope effect associated with denitrification of nitrite

The overall isotopic fractionation factor observed for denitrification of NO-2 by resting cultures and cell-free extracts of Pseudomonas stutzeri varied widely with the concentration of NO-2 and reductant. The observed isotope effect (beta obs) increased linearly with velocity when the concentration of nitrite was varied and decreased with velocity when reductant concentration was varied. At any given ratio of velocity to maximum velocity, beta obs was approximately the same in intact cells and cell free extracts. These results indicate the following: (a) neither uptake (whole cells) nor enzyme-substrate association (cell-free extracts) is the sole cause of saturation of the overall rate as [NO-2] is increased; (b) a reductive step lies beyond the initial step and at or before the first unidirectional step; and (c) in intact cells, uptake of NO-2 by the cell and egress of NO-2 from it are very rapid compared to reduction at all concentrations of NO-2. A corollary of the last conclusion is that variation of beta obs in intact cells is due entirely to variation in the relative rates of intracellular steps of the denitrification process. The linear relation between beta obs and velocity imposes constraints on any proposed mechanism of denitrification.