Role of the Surface-Exposed and Copper-Coordinating Histidine in Blue Copper Proteins:  The Electron-Transfer and Redox-Coupled Ligand Binding Properties of His117Gly Azurin

Abstract

In many reduced blue copper proteins the C-terminal surface-exposed active-site histidine protonates at low pH and dissociates from the Cu atom. In this state, the proteins exhibit high reduction potentials and low oxidation rates. In contrast, the homologous histidine (117) of azurin does not protonate. This difference has been examined by studying the electrochemical behavior of an azurin mutant in which histidine 117 is replaced by glycine to create a cavity enabling external ligands to enter the protein and coordinate to the Cu. We show that the external ligands influence the electrochemical properties of the copper site, as studied with potentiometric titrations of protein solutions and with fast-scan and low-temperature cyclic voltammetry of protein films adsorbed on graphite electrodes. The reduction potential (E 0′ ) of His117Gly azurin without external ligands is very high, at 670 ( 10 mV, but it decreases upon addition of Clor imidazole. The reduced form has little affinity for these ligands; however, under fast-scan or cryoscopic conditions (-70 °C, 70% methanol) the reduced form of the imidazole complex can be "trapped", and a reversible redox couple is established. The electrochemical kinetics of the trapped state are very fast and similar to those of wild-type (wt) azurin. The reduction potential is ∼60 mV lower than for wt azurin under identical conditions. The dissociation constant K diss of the Cu(I)-imidazole complex lies between 14 and 69 M at 20 °C, while that of the Cu(I)-Clcomplex is estimated to be as high as 10 6 M. These very low affinities show that for wt azurin the covalent link between the imidazole side chain of His117 and the protein framework is crucial for maintaining this side chain as a ligand of the Cu(I) ion.

A New Type 2 Copper Cysteinate Azurin

“…The eluted dimer fraction was colorless, compatible with the copper being in the reduced state. The copper could not be reoxidized with potassium ferricyanide to the Cu(II) form as could have been expected for a H117G azurin variant (6,9). The possibility for the dimer being in its apo form is not likely because reconstitution with copper could not be achieved.…”

“…The extinction coefficient of the absorption at 385 nm in H117G/N42C azurin has been estimated from the ratio of the absorptions at 385 nm of the Cu-H117G/N42C azurin and 280 nm of the H117G/N42C apoazurin. For the extinction coefficient at 280 nm the value of H117G apoazurin has been used (⑀ 280 ϭ 9.1 ϫ 10 3 M Ϫ1 cm Ϫ1 ) (9). Samples were measured in 20 mM HEPES, pH 7, unless stated otherwise.…”

Basic requirements for a metal-binding site in a protein: The influence of loop shortening on the cupredoxin azurin

“…Loop mutation experiments have shown that, in most cases, lengthening the ligand-containing loop lowers the His ligand pK a (9,10,12,38), whereas loop contraction increases the value (8,11,12). In AZ, the His-117 ligand does not protonate in the accessible pH range, and a pK a of Ͻ2 has been calculated (20). In the Cu(I) AZAMI loop-contraction variant, the pK a of His-115 is 5.5 [this pK a is lower than the value of 6.4 for His-96 in Cu(I) AMI] (21,38).…”

Dicopper(II) and Dicadmium(II) Complexes of Double-Tweezer Ligand with Thiopyridine Moiety

“…[1][2][3] In particular, dicopper complexes coordinated by histidine N-donor and S-containing thiolate or sulfide ligands comprise the active sites of key metalloproteins. 4,5 Accordingly, an approach to the structure and functions of the copper centers is to develop model dicopper-multidentate ligand systems incorporating N and S donors. 6 Recently, Hanton et al 7 introduced a flexible ligand with four thiopyridyl arms L [1,2,4,5-tetrakis(2-pyridylmethylsufanylmethyl)benzene] which forms a dinuclear complex with Cd(NO 3 ) 2 instead of a polymeric array due to the unusual three-layered π-stacking motif.…”

“…In this paper, we report two binuclear complexes [Cu 2 L(ClO 4 (2), which were structurally characterized by single-crystal X-ray analysis.…”