The Mo/W-bisPGD enzyme superfamily comprises a vast number of mononuclear molybdenum and tungsten enzymes that catalyze a great diversity of vital reactions in prokaryotes. In the past decades, much attention has been devoted to the immediate surroundings of the metal atom highlighting the importance of the inner coordination sphere but has failed to identify molecular determinants of the reactivity. Here, we report the mechanistic importance of a set of conserved residues that line the substrate entry tunnel in Escherichia coli nitrate reductase A (Nar), a paradigmatic enzyme of the Mo/W-bisPGD superfamily. Using mutagenesis, enzyme kinetics, electron paramagnetic resonance spectroscopy, and molecular dynamics, we unveil the pivotal role of Glu-581 motion and a number of polar residues in its close proximity in substrate affinity and proton transfer to the Mo active site. Motion of the side chain of Glu-581 exhibiting a strong acid–base cooperativity with Asp-801 and surrounded by several polar interactions controls the hydration inside the protein core, proton transfer, and substrate selectivity toward the active site. Overall, we identify an additional determinant that fine-tunes the reactivity and selectivity in Nar and propose that a gating mechanism is at play in several other members of the superfamily.