Nitrate reductases from rhizobacteria : a Nar-like membrane-bound nitrate reductase from Sinorhizobium melitoti 2011

FELIX M FERRONI; MARIA G RIVAS; MARIA ESTER LUCCA; NORA PEROTTI; ALBERTO C RIZZI; CARLOS A BRONDINO

SALTA

Workshop; SAB 2010, Workshop SeBEM, Latin American Protein Society Meeting; 2010

SAB

Nitrate reductases from rhizobacteria: a Nar-like membrane-bound nitrate reductase from Sinorhizobium meliloti 2011.Felix M. Ferronia, María G. Rivasb, María E. Luccac, Nora I. Perottic, Alberto C. Rizzia, Carlos D. Brondinoa.a Physics Department, Universidad Nacional del Litoral, Ciudad Universitaria, Paraje El Pozo, S3000ZAA, Santa Fe, Argentina.bREQUIMTE, Chemistry Department, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal.cPROIMI-CONICET Av. Belgrano y Caseros, 4000 Tucumán, Argentina.(fferroni@fbcb.unl.edu.ar)We report the purification and biochemical characterization of the respiratory membrane-bound nitrate reductase from Sinorhizobium meliloti 2011 (Sm NR). The highest levels of Sm NR activity were obtained under microaerobic conditions in presence of both nitrate and nitrite. Sm NR was obtained by treatment of disrupted spheroplasts membranes with Triton X-100 and was successfully purified after three anion exchange chromatographic steps and a final electro-elution process. Sm NR is a Mo- and Fe-containing heterodimeric protein (118 kDa and 45 kDa) and belongs to the DMSO reductase family of mononuclear Mo-proteins1. The UV-vis spectra of as-isolated and dithionite reduced Sm NR show characteristic absorption bands of iron-sulfur and heme centers. Cell growth in presence of either molybdate or tungstate indicates that molybdenum is essential for the Sm NR activity, which is in line with kinetic studies of the as-isolated enzyme that suggest that the inhibition occurs by replacement of W for Mo. Preliminary MS/MS studies show an association with respiratory nitrate reductases (Nar). This result is in agreement with the patterns of the inhibition assays.References:1. González, P.J., Correia, C., Moura, I., Brondino, C.D. and Moura, J.J.G. Journal of Inorganic Biochemistry, 100, 1015-1023, 2006.Acknowledgments: ANPCyT, CONICET and UNL.