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656 Abstracts N01 Engineering copper sites in proteins Gerard W. Canters~ Jan Coremans, Christopher Dennison, Edgar Groenen, /krnout Kalverda, Sandra Kroes, Gertie van Pouderoijen, Erik Vijgenboom Leiden Institute of Chemistry, Gorlaeus Laboratories, Einsteinweg 55, PO Box 9502, 2300 RA Leiden, The Netherlands. The properties of the Cu-sites in the so-called blue copper proteins can be engineered extensively by means of site-directed mutagenesis. Two types of modification are feasible [1]. The first is the direct replacement of one of the ligands of the Cu by another amino acid. The second consists of changing one of the ligands into an amino acid residue with a small side chain, preferably a glycine [2]. The hole thus created in the coordination shell of the Cu can be filled with externally added ligands. The newly created sites generally appear to fit into the type-I/type-2 division of Cu sites, but intermediate forms are also observed of which no equivalent exists in nature. In type-1 sites the Cu is coordinated in a trigonal-planar fashion by 2 histidines and a cysteine (N2S-coordination) with possibly one or two weakly interacting axial ligands (often a methionine sulfur among them). The type-2 sites, on the other hand most likely correspond to a more or less square planar arrangement of 4 ligands around the Cu. The intermediate forms are thought to correspond with a continuum of configurations, by which the type-1 configuration via an axial distortion, gradually transforms into an intermediate tetrahedral configuration and finally by a further distortion into a type-2 site. Another type of modification consists of changing a whole loop. In this way the Cu-site of amicyanin has been transformed, for instance, into a plastocyanin type site and into a CUA site [3]. During the course of these investigations there arose the need to quantify the axial distortion of the the site. Good criteria appear to be the rhombicitiy in the EPR spectrum, the frequencies of the prominent bands in the RR spectra and the intensity ratio of the optical bands at 450 and 600 nm. Precise information about the spin density distribution on the Cu and its ligands are obtained from W-band (100 GHz) pulsed EPR, ENDOR and ESEEM investigations of single crystals of the blue copper proteins [4]. With special techniques it appears possible to measure the NMR spectra of the oxidised forms of the blue copper proteins. The dipolar shifts of the ligand signals give direct information about the spin density on the ligands. The previously developed measures for the strength of the axial interaction parallel the more precise estimates for the Cu-S(Met) interaction obtained from the NMR investigations. [ll [21 [3] I41 Canters, G.W. et al. (1993) FEBS Lett. 325 39-48. Den Blaauwen et al. (1993) Biochemistry 32 12455-12464. Dennison, C. et al. (1995) FEBS Lett. in the press Coremans, J.W.A. et al. (1994) J. Amer. Chem. Soc. 116 3097-3101

Engineering copper sites in proteins

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656 Abstracts

N01 Engineering copper sites in proteins Gerard W. Canters ~ Jan Coremans, Christopher Dennison, Edgar Groenen, /krnout Kalverda, Sandra Kroes, Gertie van Pouderoijen, Erik Vijgenboom Leiden Institute of Chemistry, Gorlaeus Laboratories, Einsteinweg 55, PO Box 9502, 2300 RA Leiden, The Netherlands.

The properties of the Cu-sites in the so-called blue copper proteins can be engineered extensively by means of site-directed mutagenesis. Two types of modification are feasible [1]. The first is the direct replacement of one of the ligands of the Cu by another amino acid. The second consists of changing one of the ligands into an amino acid residue with a small side chain, preferably a glycine [2]. The hole thus created in the coordination shell of the Cu can be filled with externally added ligands. The newly created sites generally appear to fit into the type-I/type-2 division of Cu sites, but intermediate forms are also observed of which no equivalent exists in nature. In type-1 sites the Cu is coordinated in a trigonal-planar fashion by 2 histidines and a cysteine (N2S-coordination) with possibly one or two weakly interacting axial ligands (often a methionine sulfur among them). The type-2 sites, on the other hand most likely correspond to a more or less square planar arrangement of 4 ligands around the Cu. The intermediate forms are thought to correspond with a continuum of configurations, by which the type-1 configuration via an axial distortion, gradually transforms into an intermediate tetrahedral configuration and finally by a further distortion into a type-2 site. Another type of modification consists of changing a whole loop. In this way the Cu-site of amicyanin has been transformed, for instance, into a plastocyanin type site and into a CUA site [3]. During the course of these investigations there arose the need to quantify the axial distortion of the the site. Good criteria appear to be the rhombicitiy in the EPR spectrum, the frequencies of the prominent bands in the RR spectra and the intensity ratio of the optical bands at 450 and 600 nm. Precise information about the spin density distribution on the Cu and its ligands are obtained from W-band (100 GHz) pulsed EPR, ENDOR and ESEEM investigations of single crystals of the blue copper proteins [4]. With special techniques it appears possible to measure the NMR spectra of the oxidised forms of the blue copper proteins. The dipolar shifts of the ligand signals give direct information about the spin density on the ligands. The previously developed measures for the strength of the axial interaction parallel the more precise estimates for the Cu-S(Met) interaction obtained from the NMR investigations.

[ll [21 [3] I41

Canters, G.W. et al. (1993) FEBS Lett. 325 39-48. Den Blaauwen et al. (1993) Biochemistry 32 12455-12464. Dennison, C. et al. (1995) FEBS Lett. in the press Coremans, J.W.A. et al. (1994) J. Amer. Chem. Soc. 116 3097-3101