X-ray Structure Determination and Characterization of the Pseudomonas aeruginosa Azurin Mutant Met121Glu^,

Abstract

The Met121Glu azurin mutant has been crystallized and the structure determined at a resolution of 2.3 A. In the crystal structure a carboxyl oxygen of Met121Glu is coordinated to the metal at a distance of 2.2 A. Single-crystal resonance Raman spectroscopy was used to show that the glutamic acid residue in the copper site was in the protonated state. Titration of this residue gives rise to a number of unusual, pH-dependent properties: as the pH is increased from 4 to 8, the S(Cys)-Cu ligand-to-metal charge transfer bands are blue shifted and their intensity ratio is reversed, the EPR signal changes from type 1 copper to a new form of protein-bound copper, and the redox potential changes from 370 to 180 mV. The spectroscopic changes in this pH interval are consistent with a two-state model. From the pH dependence of the optical and EPR spectra, pKa = 5.0 for the glutamic acid in the oxidized protein was determined.

Reduction potential variations in azurin through secondary coordination sphere phenylalanine incorporations

“…Finally, the Met121Leu mutation also shifted the reduction potential ∼60-80 mV higher in each mutant (Table 2), which is consistent with reports by others and reasonable considering a stabilization of Cu(I) by the resulting trigonal ligand set [33,47,[85][86][87][88][89][90][91][92]. Overall, the Met121Leu triple Phe substituted mutant (Met121Leu + Leu33Phe/Met44Phe/ Leu86Phe azurin) showed the highest potential (503 mV vs. NHE), with a value over 140 mV higher than WT azurin.…”

De Novo Design and Characterization of Copper Metallopeptides Inspired by Native Cupredoxins

“…Cu(II) α 3 D-ChC2 exhibited two distinct intense LMCT bands at 401 nm (4429 M −1 cm −1 ) and 499 (2020 M −1 cm −1 ). This complex formed a brown-orange copper species in solution (Supporting Information, Figure 2), which has been previously observed in the Met to Glu mutant of azurin 29 and rusticyanin. 30 The R ε value is widely used to categorize native and designed CuT1 centers and determined from the ratio of the molar extinction coefficient at ~400 (±50) and ~600 (±50) nm.…”

“…The hyperfine coupling constant value of Cu(II) α 3 D-ChC2 is 130 × 10 −4 cm −1 , which is nevertheless ~30 × 10 −4 cm −1 greater than the A ∥ values of its native counterparts. Moreover, the Cu(II) spectroscopic features of Cu(II) α 3 D-ChC2 are comparable to the oxidized form of Met148Glu rusticyanin 30 and Met121Glu azurin, 49 which both displayed a brown copper species (at pH > 6) with a λ max between 396 and 416 and a second less intense band at ~570 nm, as well as A ∥ values greater than 90 × 10 −4 cm −1 . This comparison shows that the chelate-core design was able to recapitulate a mutated CuT1-like site within the α 3 D fold and provides insight into future designs for chelate-core constructs to achieve a pure CuT1 site.…”

Atx1-like chaperones and their cognate P-type ATPases: copper-binding and transfer

“…The variation in the identity of these axial ligands, and in the solvent accessibility of the copper binding sites within these proteins, tunes the copper redox potential by stabilizing the Cu(I) state to differing extents. As such, a wide range of potentials, from~+180 to +700 mV, is observed amongst blue copper proteins (Botuyan et al 1996;Hart et al 1996;Karlsson et al 1997;Malmstrom and Leckner 1998).…”