Pseudoazurin from Sinorhizobium meliloti as electron donor to copper-containing nitrite reductase: influence of the redox partner on the reduction potentials of the enzyme copper centers

FÉLIX M. FERRONI; JACOPO MARANGON; NICOLÁS I. NEUMAN; JULIO CRISTALDI; SILVINA BRAMBILLA; SERGIO A. GUERRERO; MARÍA GABRIELA RIVAS; ALBERTO C. RIZZI; CARLOS D. BRONDINO

JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY

SPRINGER

Lugar: Berlin; Año: 2014 vol. 19 p. 913 - 921

0949-8257

Pseudozurin (Paz) is the physiological electron donor to copper-containing nitrite reductase (Nir) enzyme, which catalyzes the reduction of NO2 - to NO. The reaction mechanism of Nir involves the reduction of the T1 copper electron transfer center by the external physiological electron donor, the intramolecular electron transfer reaction from T1 to T2, and the nitrite reduction at the T2 copper catalytic center. We report the cloning, expression, and characterization of Paz from Sinorhizobium meliloti 2011 (SmPaz), the ability of SmPaz as electron donor partner of SmNir, and the redox properties of the metal centers involved in the electron transfer chain. Gel filtration chromatography and SDSPAGE together with UV-vis and EPR spectroscopies revealed that as-purified SmPaz is a mononuclear copper-containing protein that presents a T1 copper site in a highly distorted tetrahedral geometry. The SmPaz/SmNir interaction investigated electrochemically showed that SmPaz serves as an efficient electron donor to SmNir. The formal reduction potentials of T1 in SmPaz and T1 and T2 in SmNir, evaluated by cyclic voltammetry, and by UVvisand EPR-mediated 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 reversed, in line with a T1?T2 electron transfer thermodynamically more favorable.