250 Spectroscopic and theoretical investigations of the geometric and electronic structures of mononuclear and binuclear copper sites in proteins help in understanding the contributions of these proteins to biological electron transfer. Spectroscopically calibrated density functional theory calculations, which give reasonable bonding descriptions in both ground- and excited-states, define the role of the protein in determining the geometric and electronic structure of the active site. Addresses Department of Chemistry, Stanford University, Stanford, CA 94305, USA *e-mail: edward.solomon@stanford.edu Current Opinion in Chemical Biology 2002, 6:250–258 1367-5931/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Published online 13th February 2002 Abbreviations B(38HF)P BP set of functionals with 38% Hartree–Fock exhange substitution B3LYP Becke’s three-parameter hybrid with Lee–Yang–Paar correlation functionals BC blue copper site BLYP Becke (1988) exchange and Lee–Yang–Parr (1988) correlation functionals BP Becke (1988) exchange and Perdew (1986) correlation functionals CT charge-transfer DFT density functional theory ENDOR electron nuclear double resonance EPR electron paramagnetic resonance ET electron transfer GC green copper site GGA generalized gradient-corrected approximation LF ligand field LT low temperature (liquid nitrogen) LUMO lowest unoccupied molecular orbital MCD magnetic circular dichroism PES potential energy surface SCF self-consistent field XAS X-ray absorption spectroscopy Xα-SW X-α scattered wave Introduction Copper-containing metalloproteins play important roles in biological electron transfer (ET) and oxygen binding, activation and reduction to water (see also Update). This Current Opinion review focuses on electronic structure/function correlations for the ET sites in mono- nuclear blue copper sites (BCs) and binuclear Cu A proteins. The geometric and electronic structures of the copper sites involved in O 2 chemistry and their relation to function have recently been reviewed [1 •• ]. Here, we focus on the unique spectral features of the ET sites, which reflect novel electronic structures, and their contributions to rapid, long-range, directional ET. Mononuclear ET sites The oxidized BC active site contains a Cu(II) ion with two normal histidines, a short equatorial cysteine and a long axial methionine ligand (Figure 1a, [2]). The overall coordination geometry of the copper site is a trigonally distorted tetrahedron. The ground-state [3] is S = 1/2 with a hole in the d x 2 –y 2 orbital from its electron paramagnetic resonance (EPR) spectrum with g || > g ⊥ > 2.00. This site exhibits a very small parallel hyperfine coupling constant (A || ) in its EPR spectrum and an intense π→Cu(II) charge- transfer (CT) band in its absorption spectrum, which strongly differ from those of normal Cu(II) complexes, such as tetragonal [CuCl 4 ] 2– . The source of these unique spectral features is the highly covalent Cu–S(Cys) π bond [3,4]. Early self-consistent field (SCF) Xα-SW (X-α scattered wave) calculations [3,4] provided general insight into the electronic structure of this site. However, these calculations gave excess ligand character in the ground-state wave function relative to experiment, corresponding to an inverted bonding situation (Figure 2). Adjustment of the Cu Xα sphere parameters to fit the experimental g-values resulted in good agreement with other spectroscopic data and a good quantitative description of the ground state. The bonding descriptions obtained by modern density functional theory (DFT) methods are very similar (RK Szilagyi, M Metz, EI Solomon, unpublished data). Generalized gradient- corrected approximation DFT (GGA DFT [5]; e.g. BP or BLYP) is too covalent (Table 1) and the ligand field (LF)- and CT-transitions are calculated at higher and lower energies, respectively, than experimentally observed. The B3LYP hybrid DFT method [5] improves the ground- and excited-state descriptions, but it is still too covalent. The covalency of the Cu–S bond can be directly quantitated experimentally by ligand (sulfur) K-edge X-ray absorption spectroscopy (XAS) and metal (copper) L-edge XAS, which utilize sulfur 1s and copper 2p core electron excitations, coupled with low-temperature magnetic circular dichroism (LT MCD) spectroscopy, which gains intensity through spin-orbit coupling between excited/excited and ground/ excited states. The theory and application of these methods have been reviewed [6 • ,7 • ]. For the BC site, metal L-edge and ligand K-edge XAS spectroscopy define 41% copper 3d [8] and 38% S(Cys) 3p character [9] in the βLUMO (lowest unoccupied molecular orbital) of the ground-state wave- function (Table 1) with strong π character. The remaining ligand contributions are derived from the two equatorial N(His) (8% by electron nuclear double resonance (ENDOR) spectroscopy [10]) and Cys β-methylene H atoms (2% by paramagnetic NMR [11]). The anisotropic covalency of the thiolate–copper bond in the BC site (Figure 1a) provides strong electronic coupling [12 •• ] Electronic structure and its relation to function in copper proteins Robert K Szilagyi and Edward I Solomon*