KINETIC AND MOLECULAR PROPERTIES OF THE COPPER-CONTAINING NITRITE REDUCTASE FROM SINORHIZOBIUM MELILOTI 2011

RIVAS MG; GUEVARA CUASAPUD, LA; DURE, ANDREA; RAMIREZ, C.S.; FERRONI FM; DALOSTO, SERGIO; GONZALEZ, PABLO; BRONDINO CD

Campinas, SP

Congreso; the 47th Annual Meeting of the Brazilian Biophysical Society; 2023

Brazilian Biophysical Society (SBBf)

Sinorhizobium meliloti 2011 is a microorganism used as bioinoculant in alfalfa seeds in which the reduction of nitrite to nitric oxide is catalyzed by a copper-containing nitrite reductase (SmNirK). SmNirK is isolated as a homotrimer with two Cu centers per monomer, one type 1 (T1Cu) and one type 2 (T2 Cu). T1Cu functions as an electron transfer center (ET) involved in intra- and interprotein ET processes, while T2Cu is the active site of the enzyme where proton-coupled nitrite reduction occurs. Both Cu sites are connected by two pathways: the shorter Cys-His bridge and the longer pathway known as the sensing loop (Figure). Our research aims to investigate the electron transfer process that occurs during the catalytic mechanism of nitrite reduction by evaluating the importance of the amino acids of the first and second T2Cu coordination spheres. Two variants were constructed for the first T2Cu coordination sphere: H172D, in which the His of the Cys-His bridge was mutated by an aspartic acid, and H342G, in which the His of the adjacent subunit was replaced by a glycine. From the second coordination sphere of T2Cu, the E315 residue was mutated by an alanine and the catalytic D134 from the sensing loop was replaced by a serine. Enzyme kinetics, thermal shift assays, UV-visible, and EPR studies were performed to evaluate the catalytic efficiency, stability and changes in reduction potential of the Cu centres. H172D is a variant that retains the ability to bind the substrate but is inactive, probably due to the loss of a hydrogen bond between H172 and C171 present in the wild-type enzyme. Replacement of H342 with the noncoordinating glycine residue results in a variant with two Cu sites, where the side-chain oxygen of the catalytic aspartic acid (D134) approaches T2 ending within bonding distance with T2Cu, and with very low enzyme activity. In the E315A variant, the interaction between the active site and the substrate is impaired, as confirmed by the observed changes in the reduction potential and kinetic parameters. Replacement of the catalytic aspartic of the sensing loop with a serine (D134S) results in a much less active enzyme, where the reduction potential of T2Cu approaches that of T1Cu. The results show how changes in residues in the first and second coordination spheres of the catalytic site cause modifications in coordination geometry, H-bonds, or reduction potential, thereby altering the electron transfer process.