Heterologous production and functional characterization of Bradyrhizobium japonicum copper-containing nitrite reductase and its physiological redox partner cytochrome c550

DURE, ANDREA; RIZZI, ALBERTO C; BRONDINO, CARLOS D.; CRISTALDI, JULIO C.; RAMIREZ, CINTIA; GONZALEZ, PABLO; FERRONI, FELIX M.; DALOSTO, SERGIO; RIVAS, MARIA GABRIELA

METALLOMICS

ROYAL SOC CHEMISTRY

Año: 2020 vol. 12 p. 2084 - 2097

1756-5901

Twodomain copper-nitrite reductases (NirK) contain two types of copper centers, oneelectron transfer (ET) center of type 1 (T1) and a catalytic site of type 2(T2). NirK activity is pH-dependent, which has been suggested to be produced bystructural modifications at high pH of some catalytically relevant residues. Tocharacterize the pH-dependent kinetic of NirK and the relevance of T1 covalencyin intraprotein ET, we studied the biochemical, electrochemical, andspectroscopic properties complemented with QM/MM calculations of Bradyrhizobium japonicum NirK (BjNirK)and of its electron donor cytochrome c550(BjCycA). BjNirK presents absorptionspectra determined mainly by a S(Cys)3pp→Cu2+ligand-to-metal charge-transfer (LMCT) transition. The enzyme shows lowactivity likely due to the higher flexibility ofa protein loop associated with BjNirK/BjCycA interaction. Nitrite is reduced athigh pH in a T1-decoupled way without T1→T2 ET in which proton delivery fornitrite reduction at T2 is maintained. Our results are analyzed in comparisonwith previous results found by us in Sinorhizobium meliloti NirK, whose mainUV-vis absorption features are determined by S(Cys)3ps/p→Cu2+ LMCT transitions.