Relation between the Structure and Spectroscopic Properties of Blue Copper Proteins

Abstract

The electronic spectra of three rhombic type 1 blue copper proteins, nitrite reductase, pseudoazurin, and cucumber basic protein, have been studied by ab initio multiconfigurational second-order perturbation theory (the CASPT2 method). The six lowest excitations have been calculated and assigned with an error of less than 1800 cm-1. The singly occupied orbital in the ground-state forms a strongly covalent antibond between the copper ion and the thiolate group of the cysteine ligand with a mixture of σ and π character. This is in contrast to the axial type 1 copper protein plastocyanin which has an almost pure Cu−SCys π interaction. The two brightest lines in the absorption spectrum originate from transitions to the corresponding σ (∼460 nm) and π (∼600 nm) bonding orbitals. The relative intensity of these two lines is determined by the character of the ground-state orbital. It is possible to obtain a structure closely similar to the one found in nitrite reductase by geometry optimizations with the hybrid density functional B3LYP method in vacuum.It is a tetragonal structure with bonds of mainly σ character to the four ligands like normal square-planar Cu(II) complexes, but the cysteine thiolate group donates much charge to the copper ion and thereby makes the structure strongly distorted toward a tetrahedron. Both this structure and a trigonal π-bonded structure, which also can be obtained for all complexes and is an excellent model of plastocyanin, are equilibrium structures (although usually not with the same ligand models). They have virtually the same energy (within ∼7 kJ/mol), and the barrier between them is low. Therefore, small differences in the structure and electrostatics of different proteins may lead to stabilization of one or the other of the structures. The results indicate that axial type 1 proteins have a trigonal structure with an almost pure Cu−SCys π bond, whereas rhombic type 1 proteins have tetragonal structures with a significant σ character in this bond. Type 1.5 and 2 copper−cysteinate proteins arise when the tetragonal structure becomes more flattened than in nitrite reductase, probably by the inclusion of stronger (type 1.5) and more (type 2) ligands.

Theoretical Study of the Electronic Spectra of Small Molecules That Incorporate Analogues of the Copper–Cysteine Bond

“…7,[14][15][16][17][18][19][20] However, there has been less attention on small molecules that incorporate the copper-cysteine bond. Ryde et al have studied the structure and reorganization energy of small models of the copper cysteine bond, including CuSH 0/+ and [Cu(imidazole-CH 3 ) 2 (SC 2 H 5 (CH 3 SC 2 H 5 )] 0/+ , using a number of theoretical methods including density functional theory (DFT) with the B3LYP exchange-correlation functional, perturbation theory and coupled cluster theory.…”

Thermodynamic equilibrium between blue and green copper sites and the role of the protein in controlling function

“…In a study on a loop mutant of Amicyanin, a more limited temperature dependence was observed and assigned to the Boltzmann population of two electronic minima (25). In density functional theory (DFT) studies with a B3LYP hybrid functional, Ryde and coworkers predicated two minima, one similar to the blue copper site and a second similar to the green copper site at comparable energy (26). However, this depends on the functional and amount of HartreeFock mixing (5).…”

Determination of the geometric structure of the metal site in a blue copper protein by paramagnetic NMR

“…The electronic structure of the blue copper site of plastocyanin has been studied extensively during the last decades both by experimental approaches (5,16,17,38) and quantum chemical calculations (5,10,(25)(26)(27)47). It was found that the unpaired electron spin density is located predominately at the atomic orbitals of the four atoms in the trigonal NNS-plane, i.e., the copper atom, the Cys S ␥ ligand, and the two aromatic His N ␦ ligands (5,16,17,(25)(26)(27)30), whereas the electron spin density at the Met S ␦ atom and the Cys H ␤ is small (26)(27)(28)38). Yet, it was found recently (31) that also the latter orbitals must be included in the description of the unpaired electron spin density to obtain a reliable prediction of the paramagnetic relaxation of the protons close to the copper site.…”

“…Because the paramagnetic restraints are derived from the relaxation rates of nuclei close to the metal site, the point dipole approximation of the electron spin fails and a description of the paramagnetic relaxation that takes the electron delocalization into account must be used (37). Therefore, the approach requires knowledge of the distribution of the unpaired electron spin, which can be obtained experimentally by x-ray absorption spectroscopy (16, 17), electron nuclear double resonance (38), paramagnetic NMR (28-31) spectroscopy, or theoretically by quantum chemical calculations (5,(25)(26)(27). The blue copper protein, plastocyanin from Anabaena variabilis (A.v.…”

“…So far, the geometric structure of the metal site in blue copper proteins (12)(13)(14) has been determined primarily by x-ray crystallography and extended x-ray absorption fine structure (3,23,24), whereas the electronic structure of the blue copper site has been determined theoretically from quantum chemical calculations (5,(25)(26)(27) and experimentally by x-ray absorption spectroscopy (16,17), and more recently by nuclear paramagnetic relaxation (28)(29)(30)(31). These structures have formed the basis for a detailed understanding of the biological function of the proteins by elucidating the interplay between the electronic and geometric structure of the metal site (8,15,(32)(33)(34).…”