Spectroscopic, Kinetic, and Electrochemical Characterization of Heterologously Expressed Wild-Type and Mutant Forms of Copper-Containing Nitrite Reductase from Rhodobacter sphaeroides 2.4.3

Abstract

We report the development of a high-yield heterologous expression system for the copper-containing nitrite reductase from a denitrifying variant of Rhodobacter sphaeroides. Typical yields of wild-type protein are 20 mg L-1, which can be fully loaded with copper. Nitrite reductase contains an unusual blue-green Type 1 copper center with a redox/electron transfer function and a nearby Type 2 center where nitrite binds and is reduced to nitric oxide. The wild-type enzyme was characterized by: (1) its blue-green Type 1 optical spectrum; (2) its EPR spectrum showing rhombic character to its Type 1 center and nitrite perturbation to its Type 2 center; (3) its 247-mV Type 1 midpoint potential which is low relative to other Type 1 centers; and (4) its kinetics as measured by both steady-state and stopped-flow methods. The Type 2 copper reduction potential as monitored by EPR in the absence of nitrite was below 200 mV so that reduction of the Type 2 center by the Type 1 center in the absence of nitrite is not energetically favored. The mutation M182T in which the methionine ligand of Type 1 copper was changed to a threonine resulted in a blue rather than blue-green Type 1 center, a midpoint potential that increased by more than 100 mV above that of the wild-type Type 1 center, and a somewhat reduced nitrite reductase activity. The blue color and midpoint potential of M182T are reminiscent of plastocyanin, but the Type 1 cupric HOMO ground-state electronic g value and copper hyperfine properties of M182T (as well as cysteine and histidine ENDOR hyperfine properties; see next paper) were unchanged from those of the blue-green native Type 1 center. His287 is a residue in the Type 2 region whose imidazole ring was thought to hydrogen bond to the Type 2 axial ligand but not directly to Type 2 copper. The mutation H287E resulted in a 100-fold loss of enzyme activity and a Type 2 EPR spectrum (as well as ENDOR spectra; see next paper) which were no longer sensitive to the presence of nitrite.

pH Dependence of Copper Geometry, Reduction Potential, and Nitrite Affinity in Nitrite Reductase

“…We were unable to identify in this way the NOO stretching vibration of the enzyme-bound nitrite between 1100 and 1000 cm Ϫ1 , since this spectral range was dominated by buffer and protein absorptions. Although previous studies have discussed the binding of nitrite inferred from a perturbation to the type 2 copper EPR signal (9,23), the FTIR spectroscopy measurements presented here provide a more direct spectroscopic identification of nitrite binding to copper, since the signal derives from the nitrite ligand. The pH dependence of the reduction of nitrite to nitric oxide shows an optimum near pH 6 (23), and the corresponding pK value for the midpoint activity is ϳ6.8.…”

“…The reduction potential of the type 2 copper site is crucial for the activity of the protein, since a lower reduction potential of the type 2 copper site relative to that of the type 1 copper site retards electron transfer. Previous measurements have led to the conclusion that the reduction potential of the type 2 copper site in RsNiR is among the lowest observed for Cu-NiRs (1,9) and that nitrite binding to the type 2 copper site is needed to shift the balance of the copper site reduction potentials so as to allow electron transfer (9). Because pH-dependent changes in geometry were observed in our structures, we determined the reduction potential of the type 2 copper site using EPR at pH 6.0 and 8.4 by recording a full redox titration under anaerobic conditions.…”

“…Table 2 summarizes the processing and refinement statistics. Redox titration in combination with EPR spectroscopy were performed essentially as described by Olesen et al (9). The concentration of type 1 copper was determined at 590 nm using an absorption coefficient of 2300 M Ϫ1 cm Ϫ1 (9).…”

“…50 ml of this buffer solution was superfused over the enzyme solution so as to ensure an excess substrate over enzyme. The buffer composition was chosen such that the results could be compared with the kinetic data on the overall reaction, obtained by Olesen et al (9) and Zhao et al (9,23). An aqueous solution of 100 mM NaNO 2 shows a broad absorption around 1237 cm Ϫ1 (bandwidth 78 cm Ϫ1 ) corresponding to an anti-symmetric stretching vibration within the V-shaped nitrite anion.…”

“…blue or green). RsNiR is a copper-containing green NiR with two peaks of equal maximum absorption at 457 and 590 nm (9). As with all other Cu-NiRs, the protein is a homotrimer with two copper atoms per monomer, ϳ12 Å apart.…”