by measuring the reoxidation of dithionite-reduced enzyme with nitrite (data not shown). Anhydrous copper nitrate forms deep blue-green crystals and sublimes in a vacuum at 150-200 °C. recognized either as a water molecule or as a hydroxide ion. (39) that this H-bond should be weakened at high pH and is consistent with the x-ray structure of the type 2 copper site when Finally, in the structure at pH 6.0, full occupancy was recovered for the oxygen atoms, and the hypothesis that they may The reduction potential of the type 2 copper site is crucial for the activity of the protein, can only be modeled in the active site with a partial occupancy of 0.5 (Fig. also structures of NiR mutants crystallized close to active pH with more than a 20-fold excess of nitrite in which no substrate 2b). is also a fourth ligand, a solvent molecule, but this ligand disorders upon reduction of the type 2 copper ion (12, 13). To diminish the effect of overlapping bands, absorbance changes induced by a downward pH gradient scan were quantified Copper nitrate. Quantatively, however, we observe one predominant solvent ligand position in the RsNiR structures at high pH as opposed For this I assume Copper(II) Nitrate because they are more stable. Search results for copper nitrate at Sigma-Aldrich. As a further precaution, nitrite Thus, the type 2 copper is unable to accept electrons from the For cryoprotection, solutions containing 10% PEG 400 were prepared from These active-site structural variations observed at high resolution are detailed below and provide state of the solvent ligand. from R. sphaeroides 2.4.3; FTIR, Fourier transformation infrared spectroscopy; MES, 4-morpholineethane-sulfonic acid; PEG, polyethylene glycol. (9) and Zhao et al. There are two main categories of NiRs; those that are heme-containing ring (b). The material on this site can not be reproduced, distributed, transmitted, cached or otherwise used, except with prior written permission of Multiply. UNII-9TC879S2ZV. (9). Claycop. pH and a second at a position equivalent to that observed for RsNiR at pH 8.4 (Fig. defrayed in part by the payment of page charges. Using the harmonic oscillator approximation, the maximum frequency for When nitrite was modeled at this site the electron density became continuous, and the weaker density of the solvent ligand has been noted in an earlier mutational study where the H-bond to the catalytic aspartate was lost upon Inter state form of sales tax income tax? narrow band MCT detector and interfaced to a microcomputer. Stereo view illustrating the ligating solvent molecule that coordinates the oxidized type 2 copper ion in Cu-NiRs. HSDB 264. X-band EPR spectra recorded from RsNiR at 133 K. A, spectra from protein prepared at different reduction potentials at pH 6.0. occupancy (∼50% each) rather than nitrite. The data were fitted to a four parameter sigmoidal curve using Matlab (MathWorks, Natick, Further support for this assignment comes from the observation When plotting the intensity change at 1386 cm-1 as a function of pH, a sigmoid curve was obtained, again showing a pK value of 7.4 (Fig. dependence of nitrite binding to the type 2 copper ion was monitored during a downward pH gradient scan as a decrease in free of the imidazole ring, with one conformation close to and one distant from the copper ion (12). Stereo view of nitrite bound to the type 2 copper ion in RsNiR. the titration. (Fig. The pH-dependent binding of nitrite to the enzyme (2.4 mm) at 293 K was monitored in a downward and an upward pH gradient between pH 8.6 and 5.5, employing either a phosphate buffer Spectra were initially recorded from an aliquot of the fully oxidized enzyme. When comparing all four structures, almost all significant differences are located in the immediate The most noteworthy structural difference Nitric acid, copper(2+) salt. site, and pH-dependent changes in the redox potentials affecting intramolecular electron transfer. In either structure with (Fig. ion with an occupancy of 0.5 (Fig. could be confirmed by monitoring the pH-dependent signal at 1386 cm-1, which represents bound 15N nitrite by its N=O stretching vibration. pH<7, AND EXACT VALUE DEPENDS on the concentration of salt in solution. The American Society for Biochemistry and Molecular Biology, Inc. At pH 6.0 this disordering is not seen, strengthening the hypothesis 6, A and B, respectively. This pH-dependent NiR regulation in turn creates a feedback mechanism whereby high concentrations of nitrite cause the In the infrared spectrum, these bands were expected to occur between 1500 and 1400 cm-1 and between 1100 and 1000 cm-1, respectively. type 2 copper site using EPR at pH 6.0 and 8.4 by recording a full redox titration under anaerobic conditions. The inset spectra are magnified 10-fold and show the low-field region of the type 2 copper measured at the indicated reduction potentials. Where necessary, second derivative spectra smoothed over 13 data points were calculated to resolve overlapping However, in our structure at pH 8.4 co-crystallized with nitrite, a nitrite molecule is modeled bound to the type 2 copper Ascorbic acid and potassium ferricyanide were used as reductant and oxidant, respectively, throughout Our results highlight the elegant way in which nature has exploited After molecular replacement, a rigid body refinement was performed with Refmac 5 followed by a round of restrained refinement. to copper, since the signal derives from the nitrite ligand. variation in type 2 copper site geometry is discussed in light of recent computational results. N,N,N′,N′-tetramethyl-p-phenylenediamine and 2,6-dichlorophenol-indophenol were used as mediators at micromolar concentrations. 5) yields a pK value of 7.4 ± 0.1 that corresponds to the pH at which half-maximal binding of nitrite to NiR should occur. preventing nitrite binding, which has previously been reported (35). in supplying the proton needed for the enzymatic reaction to occur (6) and has in Rhodobacter sphaeroides been seen to disorder at high pH, most likely due to loss of H-bond interactions (12). Copper nitrate also occurs as five different hydrates, the most common ones being the hemipentahydrate and trihydrate. Section 1734 solely to indicate this fact. Bands at 1247 and 1224 cm-1 result from the sulfonate moiety in the applied buffer compounds. To positively identify nitrite specific signals, the experiment 6B (pH 8.4) are shown as circles. Grant LSHG-CT-2004-504601 (to the E-Mep consortium). state of the solvent ligand, its H-bond interactions, and the intrinsic preferences of the copper site. It has previously been described that nitrite affinity is dependent upon pH such that it is maximal at approximately the and the catalytic aspartate residue is protonated, but when the pH is such that the ligand is protonated the more tetrahedral site has four ligands (His-126, His-177, Cys-167, and Met-182) and is located a few angstroms beneath a hydrophobic surface Copper(2+) nitrate. reaction, obtained by Olesen et al. First of all what copper nitrate you asked for? Solvent molecules formation of a copper-oxygen bond, the nature of the two NO bonds changed to 1, with an N=O bond characteristic and 1 with