Nitrite Reduction by Copper-Containing Nitrite Reductases: Relevance of the Aspartic Acid of the Sensing Loop in the Catalysis of Nitrite Reduction

GUEVARA CUASAPUD, LORIETH ALEJANDRA; RIVAS, MARIA GABRIELA; FERRONI FELIX MARTÍN; RIZZI, ALBERTO C.; GONZÁLEZ, PABLO JAVIER; BRONDINO, CARLOS DANTE

Sao Paulo

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

SBBF

INTRODUCTION: Nitrite reductases (NirKs) play a key role in the denitrification pathway catalyzing the proton-coupledone-electron reduction of nitrite to nitric oxide (NO2- + 2 H + + e - → NO + H2O). Most of NirKs are heterotrimericenzymes containing two Cu atoms per protein monomer. The electron transfer center is a type 1 (T1) copper whereasthe catalytic center is a type 2 (T2) copper. Two pathways link T1 and T2: the Cys-His bridge and the substrate sensingloop. The substrate sensing loop is thought to work as a relay triggering the T1→T2 electron flow through the Cys-Hisbridge 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, D134is not fully conserved and NirK from Thermus scotoductus presents a serine in this position. OBJECTIVES: Study therelevance of the aspartic acid residue at the substrate sensing loop in the catalytic mechanism. MATERIALS ANDMETHODS: Site directed mutagenesis, continuous kinetic assays monitored spectroscopically and UV-visible and EPRtechniques. DISCUSSION AND RESULTS: The absorption spectrum of the as purified variant showed absorptionmaxima at 585nm and 459 nm with extinction coefficient values similar to the wild-type SmNirK(ɛ585nm=3.0(4) mM -1 cm-1 and ɛ459 nm=3.4(6) mM -1 cm -1 ). Metal incorporation was a more difficult process when compared with the wild typeSmNirK and, in agreement with this, metal quantification showed 1.6(1) Cu/monomer. Although D134S is active usingthe 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 overlappedspectral components associated with T1 and T2. As expected, the g-values of the T2 copper show differences whencompared with the wild-type enzyme.Keywords: nitrite reductase, metalloenzymes, enzyme catalysis / Supported by: FONCyT (PICT-2017-2186),CONICET (PIP 112 20150100550), and CAI-D-UNL. M.G.R, P.J.G., F.M.F and C.D.B. are member of CONICET.