Predicting nitrogen and oxygen kinetic isotope effects of nitrate reduction by periplasmic dissimilatory nitrate reductase

Abstract

Abstract Kinetic isotope effect (KIE) reveals the transition state structure of an elementary reaction and can be predicted by quantum chemical calculation. Density Functional Theory calculation of an enzymatic reaction with large numbers of atoms is computationally prohibitive, especially if explicit solvent effect is considered. Cutoff method, which simplifies an entire molecule to a cluster around a target position, can simplify position-specific equilibrium isotope effect calculation for a large organic molecule. It should also be applicable to KIE calculation of an enzymatic reaction and allow us to introduce explicit solvent molecules to the system. If this treatment is feasible and trustable, it will provide an efficient method to estimate a number of KIEs produced by enzyme reactions. Obviously, its robustness must be tested. Here, using NO3− reduction by the active site of periplasmic dissimilatory nitrate reductase (Nap) in Rhodobacter sphaeroides as an example, we built 17 models to test the influence of cutoff size, implicit, and explicit solvent effects on KIE calculation. The results show that to estimate the KIE value of an enzymatic reaction accurately and efficiently, we can first simplify the reaction model to a cutoff model with 3 proximal bonds to the active position. Then, incorporating implicit-plus-explicit solvent models can simulate a reaction environment more realistically, which is necessary for accuracy. Our calculated lnKIE values for nitrate Nap reduction at 25\u202f°C are −32.4\u202f±\u202f1.8‰ for 15N and −20.9\u202f±\u202f0.4 ‰ for 18O, respectively, with a ln18KIE/ln15KIE ratio of 0.65\u202f±\u202f0.05. Although additional reservoir-transport processes need to be considered, our calculation results are consistent with calibrated isotope effects from laboratory experiments, suggesting that the transition state we calculated depicts the general reaction mechanism of NO3− reduction by Nap.