Nitrite Reductase Activity in Engineered Azurin Variants

Abstract

Nitrite reductase (NiR) activity was examined in a series of dicopper P.a. azurin variants in which a surface binding copper site was added through site-directed mutagenesis. Four variants were synthesized with copper binding motifs inspired by the catalytic type 2 copper binding sites found in the native noncoupled dinuclear copper enzymes nitrite reductase and peptidylglycine α-hydroxylating monooxygenase. The four azurin variants, denoted Az-NiR, Az-NiR3His, Az-PHM, and Az-PHM3His, maintained the azurin electron transfer copper center, with the second designed copper site located over 13 Å away and consisting of mutations Asn10His,Gln14Asp,Asn16His-azurin, Asn10His,Gln14His,Asn16His-azurin, Gln8Met,Gln14His,Asn16His-azurin, and Gln8His,Gln14His,Asn16His-azurin, respectively. UV-visible absorption spectroscopy, EPR spectroscopy, and electrochemistry of the sites demonstrate copper binding as well as interaction with small exogenous ligands. The nitrite reduction activity of the variants was determined, including the catalytic Michaelis-Menten parameters. The variants showed activity (0.34-0.59 min(-1)) that was slower than that of native NiRs but comparable to that of other model systems. There were small variations in activity of the four variants that correlated with the number of histidines in the added copper site. Catalysis was found to be reversible, with nitrite produced from NO. Reactions starting with reduced azurin variants demonstrated that electrons from both copper centers were used to reduce nitrite, although steady-state catalysis required the T2 copper center and did not require the T1 center. Finally, experiments separating rates of enzyme reduction from rates of reoxidation by nitrite demonstrated that the reaction with nitrite was rate limiting during catalysis.

Figure 1

Antiparallel β-sheet section on the surface of Az-WT where mutations were made to model the catalytic T2 Cu binding site of NiR: (upper left) Az-NiR (Asn10His, Gln14Asp, Asn16His) and Az-NiR3His (Asn10His, Gln14His, Asn16His) T2 sites; (lower left) Az-PHM (Gln8Met, Gln14His, Asn16His) and Az-PHM3His (Gln8His, Gln14His, Asn16His) T2 sites. Images made from PDB ID 4AZU.

Figure 2

UV–visible absorption spectra of the four holo-azurin variants and Az-WT in 50 mM ammonium acetate pH 5.1. Spectrum labels correspond top to bottom with the peak intensity.

Figure 3

UV–visible absorption spectra of the four T1Hg^IIT2Cu^II-azurin variants and free CuSO₄ in 50 mM ammonium acetate pH 5.1. Spectrum labels correspond top to bottom with the peak intensity.

Figure 4

UV–visible absorption spectra of the Az-NiR3His T2 Cu center with added ligands NaCl, NaNO₂, NaN₃, and imidazole. All samples are 0.25 mM in 50 mM ammonium acetate buffer pH 5.1. The top ~700 nm peak is for azide, while chloride, nitrite, and Az-NiR3His are overlapping, and imidazole is shifted to ~620 nm.

Figure 5

X-band EPR spectra (solid lines) and overlaid simulated fits (dashed lines) for (A) T1 Cu(II) only, (B) T2 Cu(II) (T1 Hg(II)), and (C) T1 Cu(II) + T2 Cu(II) azurin variants, ~1 mM in 50% glycerol and 25 mM ammonium acetate pH 5.1 buffer. WT azurin (A) and free CuSO₄ (B) in buffer are shown for comparison. The sharp derivative signal around g ≈ 2.0 is iron impurity in the EPR cavity.

Figure 6

Cyclic voltammograms for (left) T1 Cu(II) loaded azurin variants and (right) T2 Cu(II) (T1 Hg(II)) azurin variants in 50 mM ammonium acetate pH 5.1 buffer. Scan rate: 25 mV/s with PGE working electrode and plotted vs standard calomel reference electrode.

Figure 7

Az-NiR3His (100 µM) under multiple turnovers of reduction with ascorbic acid and reoxidation in the presence of 10 mM NO₂ in 20 mM phosphate buffer at pH 6.35. The arrows indicate points where 2 equiv of ascorbic acid was added. The data point at 24 h indicates full reoxidation by nitrite.

Figure 8

Michaelis–Menten plots for the azurin variants: (a) Az-NiR3His; (b) Az-PHM3His; (c) T1 Hg(II)–T2 Cu(II) Az-NiR3His; (d) Az-NiR; (e) Az-PHM.

Figure 9

Reduction of 100 µM Az-NiR3His by 0.5 mM ascorbic acid, with an exponential fit to determine the pseudo-first-order rate constant, k′.

Figure 10

Plots of the pseudo-first-order rate constants, k′, versus the square root of the ascorbic acid concentration for reduction of azurin variants: (a) Az-NiR3His; (b) Az-PHM3His; (c) Az-NiR; (d) Az-PHM.

Figure 11

Reduction of 100 µM Az-NiR3His by 1 equiv of ascorbic acid, followed by reoxidation with 5 mM NO₂⁻ at 40 min.

Figure 12

Plots of the initial reoxidation rates versus nitrite concentration for reduced azurin variants: (a) Az-NiR3His; (b) Az-PHM3His; (c) Az-NiR; (d) Az-PHM.