The flavinyl transferase ApbE of Pseudomonas stutzeri matures the NosR protein required for nitrous oxide reduction

Abstract

The copper-containing enzyme nitrous oxide reductase (N₂OR) catalyzes the transformation of nitrous oxide (N₂O) to dinitrogen (N₂) in microbial denitrification. Several accessory factors are essential for assembling the two copper sites Cu_A and Cu_Z, and for maintaining the activity. In particular, the deletion of either the transmembrane iron-sulfur flavoprotein NosR or the periplasmic protein NosX, a member of the ApbE family, abolishes N₂O respiration. Here we demonstrate through biochemical and structural studies that the ApbE protein from Pseudomonas stutzeri, where the nosX gene is absent, is a monomeric FAD-binding protein that can serve as the flavin donor for NosR maturation via covalent flavinylation of a threonine residue. The flavin transfer reaction proceeds both in vivo and in vitro to generate post-translationally modified NosR with covalently bound FMN. Only FAD can act as substrate and the reaction requires a divalent cation, preferably Mg²⁺ that was also present in the crystal structure. In addition, the reaction is species-specific to a certain extent.

Keywords: ApbE/NosX family protein; Bacterial denitrification; Flavin binding protein; Nitrous oxide reductase; X-ray crystallography.