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

Abstract

Pseudoazurin (Paz) is the physiological electron donor to copper-containing nitrite reductase (Nir), which catalyzes the reduction of NO2 (-) to NO. The Nir reaction mechanism involves the reduction of the type 1 (T1) copper electron transfer center by the external physiological electron donor, intramolecular electron transfer from the T1 copper center to the T2 copper center, and nitrite reduction at the type 2 (T2) copper catalytic center. We report the cloning, expression, and characterization of Paz from Sinorhizobium meliloti 2011 (SmPaz), the ability of SmPaz to act as an electron donor partner of S. meliloti 2011 Nir (SmNir), and the redox properties of the metal centers involved in the electron transfer chain. Gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis together with UV-vis and EPR spectroscopies revealed that as-purified SmPaz is a mononuclear copper-containing protein that has 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 the T1 copper center in SmPaz and the T1 and T2 copper centers in SmNir, evaluated by cyclic voltammetry and by UV-vis- and EPR-mediated potentiometric titrations, are against an efficient Paz T1 center to Nir T1 center to Nir T2 center electron transfer. EPR experiments proved that as a result of the SmPaz/SmNir interaction in the presence of nitrite, the order of the reduction potentials of SmNir reversed, in line with T1 center to T2 center electron transfer being thermodynamically more favorable.

Periplasmic Nicotine Dehydrogenase NdhAB Utilizes Pseudoazurin as Its Physiological Electron Acceptor in Agrobacterium tumefaciens S33

“…Its extinction coefficient was determined to be 3.58 Ϯ 0.09 mM Ϫ1 · cm Ϫ1 at 595 nm and pH 7.0 when completely oxidized, which is similar to that of Paz from P. pantotrophus (41). The determination of copper showed that 1 mM protein contained 0.88 Ϯ 0.12 mM copper atoms, indicating that per mol bound recombinant Paz, there was 1 mol Cu, which is in agreement with previous reports (42)(43)(44)(45).…”

Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods

“…SmNir was identified to be a pseudoazurin (SmPaz) containing only a type 1 copper center [21]. Catalytic activity of Nir is maximal at pH ~ 5-6 and drops rapidly at higher pH, which is accompanied with a decrease in T1-T2 electron transfer rate [10,[22][23][24].…”

“…These studies also showed that though the T2 site is distorted at pH ~ 8, the metal center retains the capability of binding nitrite, suggesting that the lack of activity at high pH is not associated with substrate binding. Furthermore, the reduction potentials measured for most as-purified Nirs are against a favorable T1→T2 electron transfer reaction, which led to hypothesize that they are modulated upon interaction with nitrite and the physiological donor to favor electron transfer [21,[25][26][27]. This means that a decrease in potential should cautiously be taken as the possible cause of catalytic activity loss at high pH and that other factors such as the pH-dependent topological characteristics of the Cys-His bridge should also be taken into consideration.…”

“…Site-directed mutagenesis of SmNir was performed by PCR using the p22SK vector as template [10] and H171D-F/H171D-R or C172D-F/C172D-R primers (see Table 1 SmPaz was produced and purified as described previously [21]. nm.…”

“…Activity screening was performed with a continuous method monitoring how dithionitereduced SmPaz is oxidized by SmNir when nitrite is added, as described elsewhere [21].…”