MAGNETIC AND OPTICAL-PROPERTIES OF COPPER-SUBSTITUTED ALCOHOL-DEHYDROGENASE - A BISTHIOLATE COPPER(II) COMPLEX

Replacement of the catalytic Zn(II) in horse liver alcohol dehydrogena se (HLADH) with copper produces a mononuclear Cu(II) chromophore with a ligand set consisting of two cysteine sulphurs, one histidine nitrog en plus one further atom. The fourth ligand to the metal ion and the c onformation of the protein may be altered by addition of exogenous lig ands and/or the cofactor NADH. Absorbance, CD, low-temperature magneti c CD (MCD) and EPR spectra are presented of copper-substituted HLADH s amples in both 'open' and 'closed' conformations and in the presence a nd absence of the exogenous ligands pyrazole and DMSO. The EPR spectra indicate a strong, predominantly axial held about the copper(II) ion with high copper-thiol (cysteine) covalence. The optical and MCD spect ra are interpreted in terms of four d-d transitions to low energy, als o reflecting the axial ligand held, and four charge-transfer transitio ns to copper(II) between 30000 and 16000 cm(-1) arising from the two c ysteine sulphur atoms which give two pairs of oppositely signed MCD C- terms. These transitions are polarized mainly in the axial plane defin ed by Cys-46, Cys-174 and His-67. The binary complex formed with pyraz ole displays quite different EPR and optical spectra which can be unde rstood in terms of a rotation of the copper hole-orbital away from the axial plane thus decreasing sharply the copper-thiol covalence. The m agneto-optical spectra in the presence and absence of DMSO are indisti nguishable.