Effects of pH variation on the catalytic activity of wild-type and D134S variant of Nitrite Reductase from Sinhorizobium meliloti

GUEVARA CUASAPUD, LA; RIVAS MG; FERRONI F; RIZZI AC; GONZÁLEZ PJ; BRONDINO CD

Rosario

Congreso; L Annual Meeting SAB; 2022

Sociedad Argentina de Biofísica

Nitrite reductases (NirKs) catalyze the one-electron reduction of nitrite to nitric oxide(NO2- + 2 H+ + e- → NO + H2O) and carry out the second step in the denitrificationpathway of the biogeochemical nitrogen cycle. Most of NirKs are heterotrimericenzymes harboring two Cu atoms per protein monomer. The electron transfer center isa type 1 copper (T1) whereas the catalytic center is a type 2 copper (T2). T1 and T2are linked by two pathways: the Cys-His bridge and the substrate sensing loop. It wasproposed that the substrate sensing loop works as a relay, triggering the T1→T2electron transfer through the Cys-His bridge when nitrite binds to the active site. Anaspartate residue (Aspcat, Asp134 in Sinhorizobium meliloti NirK numbering) presentin the sensing loop and highly conserved in most of NirK was proposed to be essentialfor catalysis, as variant D98N from Alcaligenes faecalis and D98N/A/E fromAlcaligenes xylosoxidans GIFU1051 showed extremely low catalytic rate constant.Previously, we constructed and characterized a D134S SmNirK variant, whichshowed a lower catalytic rate than the wild-type enzyme. In this work, we present adetailed study of the effects that pH has on the catalytic activity of both wild-typeand D134S SmNirK. We observed that nitrite reduction catalyzed by both wild-typeand D134S variant are active in the pH range 5 to 7, and that both enzymes areinactive at pH higher than 7. In addition, the decreasing of kcat/Km ratio at higherpH indicates a lower affinity under this condition. The pH-dependence is in line withresults reported for other NirKs, and several reasons such changes in the Cu-T2 sitegeometry, protonation state of active-site residues and solvent molecules, and/orchanges in the reduction potentials with effects on the intramolecular electrontransfer could explain the observed behavior.