Azurin and Azurin Mutants

Abstract

An overview is presented of the structural, spectroscopic, and mechanistic properties of the azurins, a sub‐class of the small blue copper proteins. Sequence information, methods to produce and purify the proteins, and structures as determined by X‐ray diffraction and nuclear magnetic resonance (NMR) are reviewed. The NMR and electron paramagnetic resonance (EPR) properties of the paramagnetic native and metal‐substituted azurins are discussed for wild type and variant (as obtained by site‐directed mutagenesis) forms. Electron transfer properties are discussed in the light of recent electron transfer theories.

Azurin as a Protein Scaffold for a Low-coordinate Nonheme Iron Site with a Small-molecule Binding Pocket

“…In the literature, copper(II) binding to apo-azurin is relatively rapid ( t 1/2 = 14 minutes with 0.01 mM apo-azurin and 100 equiv of CuSO 4 at pH = 5.0), yielding copper(II) azurin with a strong band (λ max = 625 nm with ε = 5.7 cm -1 mM -1 ) that gives the holo-protein an intense blue color. 21-23 Under our conditions of 0.1 mM apo-azurin and 10 equiv of copper (pH = 7.0), development of the characteristic 625-nm band upon addition of copper(II) to apo-azurin occurs with a half-life of less than 5 minutes and copper(II) binding to the apo-Met121Ala protein is complete in less than 1 minute. However, the addition of copper(II) to samples of each iron(II)-substituted azurin (0.1 mM) gives less than 20% copper incorporation after 2 days.…”

“…This idea is consistent with the crystal structures and spectroscopic analysis of other metal substituted azurins (Co 2+ , Ni 2+ , Zn 2+ ), which have three strongly coordinating residues (Cys112, His47, His117) as well as binding to Gly45. 21 Below, we use quantum-chemical computations to support and enrich our understanding of the coordination environment.…”

“…When titrated with copper(II) sulfate, it showed the expected 625/280 nm ratio of 0.58. 21 The buffer was exchanged into 50 mM 3-( N -morpholino)propanesulfonic acid (MOPS)) buffer (pH = 7.0) using a PD10 desalting column (GE Healthcare, Sephadex G-25 Medium, volume 13.5 mL). The zinc content of the protein was measured by atomic absorption spectroscopy.…”

Purification and properties of Sandercyanin, a blue protein secreted in the mucus of blue forms of walleye, Sander vitreus

“…They include copper proteins such as plastacyanin (Freeman and Guss 2001) and azurin (Kolczak et al 2001); crustacyanin, a caretenoprotein complex found in the lobster, Homarus gammarus, carapace (Chayen et al 2003); and BBPs found in insects (Riley et al 1984;Huber et al 1987;Saito 1998). The blue color in BBPs is due to the presence of biliverdin IXc which is formed by the enzymatic cleavage of heme by heme oxygenase.…”

Palladium(II) and Platinum(II) Bind Strongly to an Engineered Blue Copper Protein

“…Among mutations to the primary coordination sphere of the copper site, the H117G mutation is special because it is a small change that allows significant solvent access to the metal, enabling exogenous ligands to bind to copper(II) in place of the missing His-117 residue. 46,50,51 For example, addition of copper(II) and an excess of N -methylimidazole to the apo form of H117G apo-azurin results in a characteristic 630 nm absorbance band and a coordination environment similar to the wild type protein. 50 Figure 1b illustrates the binding of N -methylimidazole to the H117G copper(II) azurin.…”