INTRAMOLECULAR ELECTRON TRANSFER WITHIN BACTERIAL COPPER-CONTAINING NITRITE REDUCTASE NirK

DURÉ AB; GONZÁLEZ PJ; RIVAS MG; FERRONI FM; RIZZI AC; BRONDINO CD

Paraná

Congreso; 54th Annual Meeting Argentine Society for Biochemistry and Molecular Biology. LIV Reunion Anual Sociedad Argentina de Investigación en Bioquímica y Biología molecular.; 2018

SAIB

Cu-containing nitrite reductase (NirK) is a key enzyme of the biogeochemical N-cycle that catalyzes the conversion of nitrite (NO2-) into nitric oxide (NO), a greenhouse gas that also degrades O3 in the atmosphere. Understanding the inner working of this enzyme is fundamental. This work aims to characterize different site-directed variants that tune reduction potentials and/or the coordination environment of the Cu centers present in NirK, using kinetic and spectroscopic techniques. Site-directed variants (H171C y E315A) were obtained by standard methods. H171C was generated to directly modify the bridge that connect the two Cu ions of NirK, while E315A was produced to study how modulating the reduction potential of the active site affects electron transfer rates and the kinetics of nitrite reduction. Steady-state kinetics, UV-Vis and CWEPR spectroscopy were used to achieve those goals.Both NirK variants were recombinantly produced. Molecular are similar to those of the wildtype NirK. UV-Vis and EPR spectroscopies showed the presence of Type-1 and Type-2 Cu2+ centers as in wild type NirK, although the T2 EPR signals were slightly different when compared with that of wild-type NirK. E315A yielded a Kcat identical to the wild-type enzyme (25 s-1) but a KM 3-fold larger (60 μM). H171C showed no catalytic activity.Amino acids residues H171 and E315 do not affect quaternary structure and cofactor content. H171C variant is inactive. E315A shows a turnover number identical to that of wild-type NirK, but a KM 3-fold larger. This could mean that tuning the reduction potential of the Cu ion at the active site (T2) affects the rate of substrate binding.