An unprecedented insight into the catalytic mechanism of copper nitrite reductase from atomic-resolution and damage-free structures

Copper-containing nitrite reductases (CuNiRs), encoded by gene, are found in all kingdoms of life with only 5% of CuNiR denitrifiers having two or more copies of Recently, we have identified two copies of genes in several α-proteobacteria of the order Rhizobiales including sp. ORS 375, encoding a four-domain heme-CuNiR and the usual two-domain CuNiR ( NiR). Compared with two of the best-studied two-domain CuNiRs represented by the blue (NiR) and green (NiR) subclasses, NiR, a blue CuNiR, shows a substantially lower catalytic efficiency despite a sequence identity of ~70%. Advanced synchrotron radiation and x-ray free-electron laser are used to obtain the most accurate (atomic resolution with unrestrained SHELX refinement) and damage-free (free from radiation-induced chemistry) structures, in as-isolated, substrate-bound, and product-bound states. This combination has shed light on the protonation states of essential catalytic residues, additional reaction intermediates, and how catalytic efficiency is modulated.