Copper‐containing nitrite reductase from Pseudomonas chlororaphis DSM 50135

Abstract

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 described so 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 of the 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 NO 2 -. Electrochemical experiments show that azurin from the same organism can donate electrons to this enzyme.

EPR and redox properties of periplasmic nitrate reductase from Desulfovibrio desulfuricans ATCC 27774

“…In contrast, the nitrate Mo(V) species is stable in the range from 200 to À500 mV; therefore, it is evident that nitrate addition to the dithionite-reduced Dd NapA modifies the redox properties of the Mo center, resulting in the stable Mo(V) ion nitrate species. A substrate-dependent redox potential of the active site have been observed in Rs NapAB [32] and in the coppercontaining NR from Pseudomonas chlororaphis DSM 50135 [33]. In contrast, the redox potential determined for the FeS center is independent of the nitrate, which confirms that the previously discussed redox-potential modulation occurs only at the level of the Mo site.…”

A Nitrite Biosensor Based on Coimmobilization of Nitrite Reductase and Viologen‐Modified Polysiloxane on Glassy Carbon Electrode

“…T2 Cu is located at the bottom of a 12 deep solvent channel formed by two monomers [16], while T1 Cu is located 7 beneath a hydrophobic surface patch which is surrounded by many negatively charged residues [26], and the interatomic distance between the two Cu sites is 12.6 [23]. The redox potentials of T1 Cu of some Cu-NiRs are in the range of 40 -100 mV vs. Ag/AgCl at pH 7.0 [27], and that of Cu-NiR from Rhodobacter sphaeroides strain 2.4.3 is 50 mV vs. Ag/AgCl at pH 7.0 [21]. The NiR used in this study belongs to the copper-containing one, which catalyzes 1-electron/2-proton reduction of nitrite to nitric oxide.…”

Mo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases

“…Therefore, it was suggested that the iron-sulfur center with a positive redox potential should vary during turnover conditions to obtain an efficient electron transfer path [26,44]. This possibility was recently demonstrated in a copper-containing nitrite reductase from Pseudomonas chlororaphis DSM 50135, in which the binding of the substrate to the active center modulates the rate of intramolecular electron transfer, changing the values of the redox potentials of the redox centers [45]. Electrochemical studies on R. sphaeroides Nap show that the kinetics of substrate binding favors intramolecular electron transfer [46].…”