Magnetic and optical properties of copper-substituted alcohol dehydrogenase: a bisthiolate copper (II) complex

Abstract

Replacement of the catalytic Zn(II) in horse liver alcohol dehydrogenase (HLADH) with copper produces a mononuclear Cu(II) chromophore with a ligand set consisting of two cysteine sulphurs, one histidine nitrogen plus one further atom. The fourth ligand to the metal ion and the conformation of the protein may be altered by addition of exogenous ligands and/or the cofactor NADH. Absorbance, CD, low-temperature magnetic CD (MCD) and EPR spectra are presented of copper-substituted HLADH samples in both 'open' and 'closed' conformations and in the presence and absence of the exogenous ligands pyrazole and DMSO. The EPR spectra indicate a strong, predominantly axial field about the copper(II) ion with high copper-thiol (cysteine) covalence. The optical and MCD spectra are interpreted in terms of four d-d transitions to low energy, also reflecting the axial ligand field, and four charge-transfer transitions to copper(II) between 30000 and 16000 cm-1 arising from the two cysteine sulphur atoms which give two pairs of oppositely signed MCD C-terms. These transitions are polarized mainly in the axial plane defined by Cys-46, Cys-174 and His-67. The binary complex formed with pyrazole displays quite different EPR and optical spectra which can be understood in terms of a rotation of the copper hole-orbital away from the axial plane thus decreasing sharply the copper-thiol covalence. The magneto-optical spectra in the presence and absence of DMSO are indistinguishable.

Copper A of Cytochrome c Oxidase, A Novel, Long‐Embattled, Biological Electron‐Transfer Site

“…Such a site was available in horse liver alcohol dehydrogenase from which the natural metal in the catalytic site, Zn, had been removed and replaced by Cu. This site did not show the strong MCD features at 480 and 520 nm typical of all Cu, sites [54]. Note also that the Chan model contradicts the results from the study on the linear electric field effect by Mims et al 1491.…”

“…In 1980, Mims et al [49] concluded from spin-echo studies and from the observation of the linear electric field effect in spin-echo EPR that 'The results indicate that the symmetry is not tetrahedral and that the ligation pattern is unlike that for either type 1 or type 2 Cu in other Cu proteins.' In order to obtain more definitive information on the ligands of Cu,, Chan, Scholes and their associates [50] purified COX from yeast auxotrophs, which had been supplemented with [1,3- [54]. Note also that the Chan model contradicts the results from the study on the linear electric field effect by Mims et al 1491.…”

Binuclear CuA Formation in Biosynthetic Models of CuA in Azurin Proceeds via a Novel Cu(Cys)2His Mononuclear Copper Intermediate

“…The electronic absorption data of the Glu114XCu A Az variants indicate two sharp peaks in the 410–600 nm region and a broad feature at ~700–800 nm, similar to the pyrazole-substituted HLADH, which displays two peaks at ~400–500 nm, and a broad feature ~600–800 nm. 60–62 The EPR results indicate the presence of mononuclear T2Cu species with thiolate ligands. In all the variants, the EPR data suggest two mononuclear Cu complexes with minor differences in ligand coordination/geometry.…”

“…Although both the g z and A z values are higher than the I x2 of Glu114GlyCu A Az, these parameters are similar to the Cu(Cys) 2 (His)L (L=pyrazole, NADH) complex of HLADH ( g z =2.200, A z =115 × 10 −4 cm −1 ). 60–62 Based on this similarity, we assign the I x2 of Glu114GlnCu A Az as having a similar Cu(Cys) 2 (His)L (L=weak axial ligand) ligation/geometry.…”

Cu(II)-amino acid–CaAl-layered double hydroxide complexes, recyclable, efficient catalysts in various oxidative transformations

“…The fifth and sixth positions are probably filled by water molecules coordinated through their oxygen atoms [18,19]. For the cysteinate-containing material the values of the above parameters indicate N amino N amino S thiolate S thiolate tetragonally-shaped coordination environment [20,21] with two molecules of water coordinated through their oxygen atoms in the fifth and sixth position.…”