Cytochrome c nitrite reductase from the bacterium Geobacter lovleyi represents a new NrfA subclass

Abstract

Cytochrome <i>c</i> nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium <i>Geobacter lovleyi</i> has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized <i>G. lovleyi</i> NrfA. We observed that the enzyme crystallizes as a dimer but remains monomeric in solution. Importantly, its crystal structure at 2.55-Å resolution revealed the presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes. The presence of EDTA did not affect the activity of <i>G. lovleyi</i> NrfA, and site-directed mutagenesis of this arginine reduced enzymatic activity to <3% of the WT levels. Phylogenetic analysis revealed four separate emergences of Arg-containing NrfA enzymes. Thus, the Ca²⁺-independent, Arg-containing NrfA from <i>G. lovleyi</i> represents a new subclass of cytochrome <i>c</i> nitrite reductase. Most genera from the exclusive clades of Arg-containing NrfA proteins are also represented in clades containing Ca²⁺-dependent enzymes, suggesting convergent evolution.