High-Field NMR Studies of Oxidized Blue Copper Proteins:  The Case of Spinach Plastocyanin

Abstract

The 800-MHz 1H NMR spectra of oxidized plastocyanin from spinach are here reported. All hyperfine-shifted signals have been assigned through saturation transfer with the reduced diamagnetic species. To detect the copper(II)-bound cysteine β-CH2 signals, a technique has been applied which is based on irradiation of regions where such signals are expected but not detected, and the corresponding saturation transfer on the reduced species is observed. At the end, a full spectrum is reconstructed which permits, for the first time, the complete 1H NMR signal assignment of an oxidized blue copper protein. These data are discussed in terms of the factors affecting the line width as related to the electronic and geometric structure of the metal center. A Karplus-type relationship is proposed between the contact shift of the Cys-84 β-CH2 protons and the Cu−S−C−Hβ dihedral angle.

Sulfur K-Edge XAS and DFT Calculations on P450 Model Complexes: Effects of Hydrogen Bonding on Electronic Structure and Redox Potentials

“…Similar covalent contributions in H-bonding were observed in NMR experiments on the blue Cu protein plastocyanin and in models of rubredoxin. 51,52 Note that this small covalent interaction can make an important contribution to H DA for efficient electron transfer.…”

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

“…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.…”

“…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.…”

“…[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. 8,10,11,51 The large Cys C β proton shift range should thus be a more reliable structural probe.…”

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

“…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).…”