Nitrite reductase from Sinorhizobium meliloti 2011: a Cu-containing enzyme that catalyzes the reduction of nitrite to nitric oxide

JULIO C. CRISTALDI; FÉLIX M. FERRONI; NICOLÁS I. NEUMAN; PABLO J. GONZÁLEZ; ALBERTO C. RIZZI; MARÍA G. RIVAS; CARLOS D. BRONDINO

Chascomús

Encuentro; IV LABIC Fourth Latin American Meeting on Biological Inorganic Chemistry V WOQUIBIO Fifth Workshop on Bioinorganic Chemistry; 2014

UBA, UNR, CONICET, AGENCIA, Workshop Argentino de Química Bioinorgánica

Session I - Metalloproteins/Metalloenzymes P19.-Nitrite reductase from Sinorhizobium meliloti 2011: a Cu-containing enzyme that catalyzes the reduction of nitrite to nitric oxide Julio C. Cristaldi, Felix M. Ferroni, Nicolas I. Neuman, Pablo J. Gonzalez, Alberto C. Rizzi, Maria G. Rivas and Carlos D. Brondino Departamento de Física, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina juliocristaldi@gmail.com INTRODUCTION Copper-containing nitrite reductase (Nir) is a key enzyme in the dissimilatory reduction of nitrate to dinitrogen in denitrifying bacteria. The Nir from Sinorhizobium meliloti 2011 (SmNir), a rhizobia bacterium that lives in root nodules of legumes and is widely used in agriculture as fertilizers1, is a homotrimeric protein that contains two Cu centers, one of type 1 (T1; also blue copper) and other of type 2 (T2; also normal copper). The enzyme catalyzes the reduction of NO2 - to NO (Eo? = 370 mV) through a catalytic mechanism that involves the binding of nitrite to T2 and the subsequent reduction to NO by reducing equivalents given by an external physiological electron donor through the T1 center (Figure 1)2,3 . Figure 1: T1 and T2 centers in SmNir. We report here the characterization of the metal centers involved in the electron transfer chain of SmNir. We also analyze the role of the physiological electron donor of SmNir, a copper-containing pseudoazurin (SmPaz) in modulating the reduction potentials of the copper centers in SmNir. EXPERIMENTAL METHODS SmPaz and SmNir were overexpressed and purified as explained elsewhere4. To characterize the electron transfer chain involved in nitrite reduction, the formal reduction potentials of the T1 copper center in SmPaz and the T1 and T2 copper centers in SmNir were evaluated by cyclic voltammetry and by UV? vis- and EPR-mediated potentiometric titrations. RESULTS AND DISCUSSION Electrochemistry The formal reduction potential of SmPaz was determined to be Eo´= 278 ± 6 mV vs SHE. The kinetic properties of the couple SmNir/SmPaz were evaluated varying the nitrite concentration. The increase of substrate concentration causes an increase of the catalytic current until saturation, indicating that the catalytic current resembles the rate of catalysis predicted by a Michaelis-Menten model (Km app = 100 ± 8 μM). This value is lower than that reported previously for SmNir (Km = 550 ± 60 μM), when methyl viologen was used as electron donor 1. UV-vis and EPR-mediated redox titrations UV-vis redox titrations yielded Eo´ = 226 ± 3 mV with n = 1.0 for the reduction potential of T1 of SmPaz. EPR redox titrations yielded Eo´= 224 ± 4 mV for T1 and Eo´ = 108 ± 5 mV for T2 of SmNir. SmNir reduction potential modulation by SmPaz. We performed EPR studies on a solution containing both proteins to evaluate possible changes in the EPR signals of SmNir in the presence of SmPaz under different experimental conditions (Figure 2). Figure 2: EPR spectra of a) SmNir/SmPaz mixture, b) dithionite reduced, c) nitrite-reacted SmNir, d) nitrite-reacted SmNir/SmPaz mixture. The inset shows the time variation of the absorbance at 597 nm of dithionite-reduced SmPaz. A comparison between spectra c and d shows clearly that the SmPaz and SmNir interaction upon nitrite addition changes the relative values of the T1 and T2 reduction potentials in SmNir, i.e. Eº´T1 < Eº´T2, as the intensity of the T1 EPR signal is higher than that of T2. This result might be indicating that the shift of the reduction potentials in SmNir is a consequence of the SmPaz/SmNir interaction. CONCLUSION The significant decrease in Km when using SmPaz as redox partner, together with the fact that the genes coding for both proteins are simultaneously expressed under denitrifying conditions, strongly suggest that SmPaz is the physiological electron donor of SmNir The formal reduction potentials of T1 in SmPaz and T1 and T2 in SmNir, evaluated by cyclic voltammetry, and by UV-vis- and EPRmediated potentiometric titrations, are against an efficient electron transfer T1Paz→T1Nir→T2Nir. EPR experiments proved that as result of the interaction SmPaz/SmNir in the presence of nitrite, the order of the reduction potentials of SmNir is reversed, in line with a T1→T2 electron transfer thermodynamically more favorable. Site directed mutagenesis studies of amino acids involved in the putative electron transfer chain Cys136-His135 of SmNir (Figure 1) are being carried out to have insight on the electron transfer triggered by the interaction SmNir- SmPaz. REFERENCES 1. Ferroni F. M. et al., J. Inorg. Biochem. (2012),114:8-14 2. Zumft W. G., Microbiol. Mol. Biol. R. (1997),61:533-616 3. Averill B. A., Chem. Rev. (1996),96:2951- 2964 4. Ferroni F. M. et al., J. Biol. Inorg. Chem. (2014), in the press. ACKNOWLEDGMENTS We thank FONCYT, CONICET, and CAI+DUNL for financial support. NIN thanks CONICET and JCC thanks FONCYT for fellowship grants. PJG, MGR, FMF, and CDB are members of CONICET-Argentina.