Metal−Ligand Interplay in Blue Copper Proteins Studied by ¹H NMR Spectroscopy:  Cu(II)−Pseudoazurin and Cu(II)−Rusticyanin

Abstract

The blue copper proteins (BCPs), pseudoazurin from Achromobacter cycloclastes and rusticyanin from Thiobacillus ferrooxidans, have been investigated by (1)H NMR at a magnetic field of 18.8 T. Hyperfine shifts of the protons belonging to the coordinated ligands have been identified by exchange spectroscopy, including the indirect detection for those resonances that cannot be directly observed (the beta-CH(2) of the Cys ligand, and the NH amide hydrogen bonded to the S(gamma)(Cys) atom). These data reveal that the Cu(II)-Cys interaction in pseudoazurin and rusticyanin is weakened compared to that in classic blue sites (plastocyanin and azurin). This weakening is not induced by a stronger interaction with the axial ligand, as found in stellacyanin, but might be determined by the protein folding around the metal site. The average chemical shift of the beta-CH(2) Cys ligand in all BCPs can be correlated to geometric factors of the metal site (the Cu-S(gamma)(Cys) distance and the angle between the CuN(His)N(His) plane and the Cu-S(gamma)(Cys) vector). It is concluded that the degree of tetragonal distortion is not necessarily related to the strength of the Cu(II)-S(gamma)(Cys) bond. The copper-His interaction is similar in all BCPs, even for the solvent-exposed His ligand. It is proposed that the copper xy magnetic axes in blue sites are determined by subtle geometrical differences, particularly the orientation of the His ligands. Finally, the observed chemical shifts for beta-CH(2) Cys and Ser NH protons in rusticyanin suggest that a less negative charge at the sulfur atom could contribute to the high redox potential (680 mV) of this protein.

NMR Hyperfine Shifts in Blue Copper Proteins: A Quantum Chemical Investigation

“…As compared with the BCPs discussed above, Rc has several unusual features, including the highest redox potential (680 mV) among BCPs, 32 and the smallest Cys C β proton NMR hyperfine shifts 11 (Table 3). All eight X-ray structures (PDB #: 1RCY, 2CAK, 1A8Z, 1A3Z, 2CAL, 1E30, 1GY2, and 1GY1) for wild-type or mutated Rc show that Asn80 is hydrogenbonded to the N-terminal His (His85), but there are two possible bonding modes.…”

“…For instance, as shown in Table 3, for the conserved Asn residue in Az, Pc, Pa, St, and Am, the amide proton and the Cα proton were consistently predicted to have negative and positive hyperfine shifts, respectively, as observed experimentally. [8][9][10][11] The Cys C α protons in these proteins were predicted to have a negative hyperfine shift, due to their proximity to the large positive spin densities of the Cys C β protons. 10 Hyperfine shift predictions for His protons are generally good, except for the C δ2 protons, where the hyperfine shift range is small, as it is with many other residues, and in some cases, assignments are uncertain.…”

“…10 Hyperfine shift predictions for His protons are generally good, except for the C δ2 protons, where the hyperfine shift range is small, as it is with many other residues, and in some cases, assignments are uncertain. 8,10,11,51 The large Cys C β proton shift range should thus be a more reliable structural probe. In fact, as shown in Table 3, the Cys C β proton NMR hyperfine shifts have large differences between the small Calc3 and large Calc7 models, while the other proton shifts between these two models are generally not very different.…”

“…This suggested that it might be possible to use these optimized geometric properties to predict shifts, using the empirical approach described previously. 11 The properties of interest are the displacement of Cu from the N His -CuN His plane (ΔCu), 57 the angle between the N His CuN His and S Cys CuS Met (O Gln ) planes (ϕ); 58,59 and the formed by the N His CuN His plane and Cu-S Cys vector (α). As observed previously, 11 the average experimental Cys C β proton hyperfine shift is not correlated with R cu-s Cys , ΔCu, ϕ, or α alone.…”

“…11 The properties of interest are the displacement of Cu from the N His -CuN His plane (ΔCu), 57 the angle between the N His CuN His and S Cys CuS Met (O Gln ) planes (ϕ); 58,59 and the formed by the N His CuN His plane and Cu-S Cys vector (α). As observed previously, 11 the average experimental Cys C β proton hyperfine shift is not correlated with R cu-s Cys , ΔCu, ϕ, or α alone. However, the following relationship using a combination of R cu-s Cys and was α recently found to give good predictions of the data: 11…”