Reassessing the Strategies for Trapping Catalytic Intermediates during Nitrate Reductase Turnover

We examined the kinetics of nitrate reduction by periplasmic nitrate reductase (Nap) by using protein film voltammetry and Solution assays. We demonstrate that, under turnover conditions, the enzyme exists as a mixture of active and inactive forms which interconvert oil a time scale that is Much slower than turnover. The dead-end species accumulates under mildly reducing conditions and at high nitrate concentration, resulting in Substrate inhibition and ill all uncommon hysteresis in the voltammetric signature. Solution assays with two electron donors having different reduction potentials fully support the electrochemical results. This illustrates the consequences of the high flexibility of the active site molybdenum coordination sphere and questions the conclusions from earlier studies ill Which attempts were made to trap catalytic intermediates of Nap in experiments carried Out Under turnover conditions at very high substrate concentration.