Copper-containing nitrite reductase from Pseudomonas chlororaphis DSM 50135: evidence for modulation of the rate of intramolecular electron transfer through nitrite binding to the T2Cu centre

D. PINHO; S. BESSON; C. D. BRONDINO; B. DE CASTRO; I. MOURA

EUROPEAN JOURNAL OF BIOCHEMISTRY

Blackwell Publishing LTD

Año: 2004 vol. 271 p. 2361 - 2369

0014-2956

The nitrite reductase (Nir) isolated from Pseudomonas chlororaphis DSM 50135 is a blue enzyme, with type 1 and type 2 copper centers, as in all copper-containing Nirs describedso far. For the first time, a direct determination of the reduction potentials of both copper centers in a Cu-Nir was performed: type 2 copper (T2Cu), 172 mV and type 1 copper (T1Cu), 298 mV at pH 7.6. Although the obtained values seem to be inconsistent with the established electrontransfer mechanism, EPR data indicate that the binding of nitrite to the T2Cu center increases its potential, favoring the electron-transfer process. Analysis of the EPR spectrum ofthe turnover form of the enzyme also suggests that the electron-transfer process between T1Cu and T2Cu is the fastest of the three redox processes involved in the catalysis: (a) reduction of T1Cu; (b) oxidation of T1Cu by T2Cu; and (c) reoxidation of T2Cu by NO2–. Electrochemical experiments showthat azurin from the same organism can donate electrons to this enzyme.