PNNL

Abstract

The microbial enzyme nitrogenase catalyzes the MgATP-dependent reduction of N2 to 2 NH3, a transformation central to the global nitrogen cycle. While the canonical Thorneley–Lowe (TL) kinetic model has long served as a mechanistic framework, it does not incorporate several recent insights. Here, we present an updated kinetic model for Mo-nitrogenase in which ET from the reduced Fe protein to the FeMo-cofactor is gated by MgATP-dependent conformational transitions and can be described as a probabilistic event. This framework quantitatively reproduces steady-state product formation rates across a broad range of experimental conditions, yielding revised estimates for key rate constants. We demonstrate that under N2 turnover, the probability of productive electron transfer (ET) decreases to ~40%, resulting in a significant fraction of Fe protein cycles that are unproductive for electron delivery. This mechanistic feature explains the observed rate limitation in N2 reduction and implies a revised minimum energetic cost of about 25 MgATP per N2 reduced. By integrating conformational dynamics and probabilistic ET into a unified kinetic framework, the model provides a more complete and adaptable foundation for understanding nitrogenase efficiency and regulation.