~ Pergamon 0277-5387(95)00028-3 Polyhedron Vol. 14, No. 15 16, pp. 2063- 2070, 1995 Copyright 73 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 027%5387/95 $9.50+0.00 MODELLING THE BLUE PROTEIN ACTIVE SITES: SYNTHESIS AND CHARACTERIZATION OF CuN2S2 COMPLEXES SHOWING RHOMBIC EPR SPECTRA AND HIGH CulI/CuI POTENTIAL SUBRATA MANDAL, RAMESHWER SHUKLA and PARIMAL K. BHARADWAJ* Department of Chemistry, Indian Institute of Technology, Kanpur, 208016, India (Received 11 October 1994 ; accepted 3 January 1995) Abstract--Two new tetradentate ligands have been synthesized by Schiff base condensation of diisobutyraldehyde disulphide with 2-mercaptoethylamine (L 1) and 2-aminothiophenol (L 2) respectively and then reducing the imine linkages with NaBH4 in refluxing methanol. In the free ligands the thiolate sulphur is protected with tertiary butyl groups which are cleaved in the presence of Cun-salts to give neutral CuN2S2 complexes. The copper com- plexes show ligand field transitions at 815 and 760 nm at room temperature which are independent of the solvents used and are consistent with a pseudotetrahedral coordination around the Cu n ion. The EPR spectrum of the aliphatic thiolate in MeCN glass shows significant rhombic splitting (gx-gy = 0.09 and Ax-A, = 60 x 10 -4 cm-l) attributable to dz2 mixing into the ground state wavefunction. For the aromatic thiolate complex, however, the EPR spectrum was not well resolved although the rhombic nature of the spectrum could easily be observed. Both the complexes exhibit well-defined cyclic responses in their cyclic voltammograms at RT and in acetonitrile for the CuI1/Cu ~ couple with E1/2 = 0.5 V vs SCE. This high positive value for the redox couple is also consistent with a coordination geometry much distorted from planarity. The active sites of the blue proteins which contain copper in distorted geometries exhibit Cu"/Cu ~potential in the range 300-800 mV vs NHE at pH = 7.0. Ligation of thiolate sulphur to copper at the active sites of quite a number of copper proteins has been established. CuH-thiolate bonding has been shown to be present by X-ray crystallography in the case of single copper proteins like plastocyanin, azurin, 2 pseudoazurin 3 and basic blue protein from cucum- ber n and in a multicopper protein, ascorbate oxidase. 5 A preliminary structure is also available 6 on the bacterial blue protein, amicyanin from Thiobacillus versutur. In the case of other single copper blue proteins like stellacyanin, umecyanin, rusticyanin or multicopper oxidases like tree and fungal laccases, ceruloplasmin etc., the presence of copper-sulphur bonding has been established by various spectroscopic studies. 7 The CUA site in cyto- * Author to whom correspondence should be addressed. chrome c oxidase also contains* two cysteine thio- lares bonded to Cu n. Moreover, for Cull-substituted proteins like horse liver alcohol dehydrogenase, this bonding is also present 9 as inferred from various spectroscopic investigations. A major reason for the peculiar spectroscopic behaviour exhibited by these proteins is due to the presence of strong CuII-thiolate bonding. 1° Hence the bioinorganic scope of this bonding is quite con- siderable. Copper blue proteins, in particular, have attracted a lot of attention by chemists who are involved in synthesizing electronic structural ana- logues of the type 1 site which is intrinsic in nature.ll The electronic spectrum of a blue site is dominated by an intense absorption band centred around 600 nm (molar Cu absorptivity lies in the range 2000- 6000) attributable to the a (thiolate) --* Cu" LMCT transition. There have been continuous efforts to 2063