NITRITE REDUCTION BY COPPER-CONTAINING NITRITE REDUCTASES: RELEVANCE OF THE ASPARTIC ACID OF THE SENSING LOOP IN THE CATALYSIS OF NITRITE REDUCTION

L.A. GUEVARA; M.G. RIVAS; F.M. FERRONI; A.C. RIZZI; P.J. GONZÁLEZ; C.D. BRONDINO

Aguas de Lindoia

Congreso; 51th Annual Meeting of the Brazilian Society of Biochemistry and Molecular Biology (SBBq)/46th Congress of the Brazilian Society of Biophysics (SBBf) / LAFESB; 2022

Brazilian Society of Biochemistry and Molecular Biology (SBBq) and Brazilian Society of Biophysics (SBBf)

INTRODUCTION. Nitrite reductases (NirKs) play a key role in the denitrification pathway catalyzing the proton-coupled one-electron reduction of nitrite to nitric (NO2- + 2 H+ + e- → NO + H2O). Most of NirKs are heterotrimeric enzymes containing two Cu atoms per protein monomer. The electron transfer center is a type 1 (T1) copper whereas the catalytic center is a type 2 (T2) copper. Two pathways link T1 and T2: the Cys-His bridge and the substrate sensing loop. The substrate sensing loop is thought to work as a relay triggering the T1→T2 electron flow through the Cys-His bridge when the substrate is bound to the catalytic center. An aspartic acid (D134, Sinhorizobium meliloti NirK –SmNirK- numbering) present in the sensing loop was proposed to be essential in the catalysis process. However, D134 is not fully conserved and NirK from Thermus scotoductus presents a serine in this position. SUBJECT. Study the relevance of the aspartic acid residue at the substrate sensing loop in the catalytic mechanism. METHODOLOGY. Site directed mutagenesis, continuous kinetic assays monitored spectroscopically and UV-visible and EPR techniques. RESULTS AND DISCUSSION. The absorption spectrum of the as purified variant showed absorption maxima at 585nm and 459 nm with extinction coefficient values similar to the wild-type enzyme (ɛ585nm=3.0(4) mM-1cm-1 and ɛ459 nm=3.4(6) mM-1cm-1). Metal incorporation was a more difficult process when compared with the wild type SmNirK and, in agreement with this, metal quantification showed 1.6(1) Cu/monomer. Although D134S is active using the physiological electron donor (pseudoazurin), the kcat and Km values decrease 240-fold and 10-fold, respectively, when compared with the wild-type SmNirK. D134S EPR signal of the as isolated enzyme showed two overlapped spectral components associated with T1 and T2. As expected, the g-values of the T2 copper show differences when compared with the wild-type enzyme.