DECIPHERING THE CONTRIBUTION OF TYPE 1 COPPER CENTERS TO THE BIOPHYSICAL PROPERTIES ON A THREE DOMAIN NITRITE REDUCTASE

Copper-containing nitrite reductases (NirKs) catalyze the reduction of nitrite (NO₂^-) to gaseous nitric oxide (NO). This highly conserved family of enzymes is widespread in nature and plays a major role in the biogeochemical nitrogen cycle. NirKs have a homotrimeric physiological structure that contains two types of copper centers: an electron-accepting type-1 copper (T1Cu) site and a catalytic type-2 copper (T2Cu) center. However, the NirK recently isolated from Thermus scotoductus SA-01 (TsNirK) has an additional C-terminal tethered cupredoxin domain (Δ308N) fused to its core structure. Additionally, the T1Cu from the main domain (T1Cu_N) exhibits a unique coordination pattern that has only been observed previously in stellacyanins (Gln ligand). The extra domain contains a T1Cu_C that serves as an additional electron transfer unit. Through recombinant overexpression, we obtained TsNirK and its truncated ∆308N form. Here, we present the biophysical characterization of these two proteins using UV-Vis, EPR, and resonance Raman spectroscopies, complemented by bioinformatic modeling. Samples were analyzed at high (10.0) and low (6.0) pH values to evaluate whether there are variations in coordination properties. This approach enables us to describe the contribution of each T1Cu center to the overall structure and the significance of the tether domain in the catalytic steps.

This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, Argentina, Project PICT-SERIE A-I-GRF-00522)