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,Asn16-His-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.

Redox Characterization of Electrode-Immobilized Bacterial Microcompartment Shell Proteins Engineered To Bind Metal Centers

“…The 3His variants and WT in the presence of 1.0 equivalence of Cu displayed a weak absorption band at ∼530 nm ( E 530 nm = 79–113 M –1 cm –1 ), which represents the typical d–d absorption profile characterized in Type II copper complexes containing N only or N/O ligands . The Cu d–d profile of the 3His variants is comparable to a small molecule compound composed of a Cu–N x complex, with a distorted planar geometry. , When compared to its native or designed counterparts, d–d absorption band of the 3His variants, which was examined at a neutral pH, is blue-shifted by 70 nm.…”

“…For example, the metal coordination site of the Cu-containing redox protein azurin was modified to shift the formal potential over a 0.7 V range when only using Cu and over a 2 V range when Ni or Cu was bound into the mutated coordination sites . Additionally, engineering a second copper binding site into an azurin protein to facilitate nitrite reduction resulted in a formal potential that was shifted by approximately 20 mV depending on the coordination environment . We note that the proteins used in these previous experiments were redox active prior to modification.…”

“…50 Additionally, engineering a second copper binding site into an azurin protein to facilitate nitrite reduction resulted in a formal potential that was shifted by approximately 20 mV depending on the coordination environment. 44 We note that the proteins used in these previous experiments were redox active prior to modification. Binding Cu on the convex side of the BMC-T1 HO protein (S56H and A157H) resulted in similar formal potentials (208 ± 7 and 213 ± 5 mV vs SHE) that were significantly lower than the variants with Cu bound to the concave side (S55H and G155H; E°′ = 256 ± 6 and 229 ± 8 mV vs SHE, respectively).…”

“…41 The Cu d−d profile of the 3His variants is comparable to a small molecule compound composed of a Cu−N x complex, with a distorted planar geometry. 42,43 When compared to its native or designed 44 counterparts, d−d absorption band of the 3His variants, which was examined at a neutral pH, is blue-shifted by 70 nm.…”

Proton-Coupled-Electron Transfer Enhances the Electrocatalytic Reduction of Nitrite to NO in a Bioinspired Copper Complex

“…In the natural CuNIR class of enzymes, an aspartate residue sits in close proximity to the bound nitrite and helps to mediate proton transfer to one of the nitrite oxygens, which is eventually lost as water. 5,10 The importance of nearby proton-donating residues in the mechanism of nitrite reduction by CuNIR has been highlighted by recent crystallographic 57 and site-directed mutagenesis 58 studies on the relevant enzymes. There have also been three seminal papers in recent years showcasing the importance of hydrogen bonding in the secondary coordination sphere of synthetic systems for nitrite reduction based on both iron 59,60 and copper, 61 although none of these systems were able to demonstrate catalytic turnover.…”