Structural Information through NMR Hyperfine Shifts in Blue Copper Proteins

Abstract

The oxidized blue copper proteins azurin and stellacyanin have been investigated through 1H NMR at 800 MHz and the results compared with those for plastocyanin (Bertini, I.; Ciurli, S.; Dikiy, A.; Gasanov, R.; Luchinat, C.; Martini, G.; Safarov, N. J. Am. Chem. Soc. 1999, 121, 2037). By exploiting saturation transfer between the oxidized and the reduced forms, all the hyperfine shifted signals can be assigned, including the β-CH2 protons of the coordinated cysteines, which are so broad not to be detected under direct observation. Both hyperfine shifts and line widths of the latter signals differ dramatically from one protein to another: average hyperfine shifts of about 850, 600, and 400 ppm and average line widths of 1.2, 0.45, and 0.25 MHz are observed for azurin, plastocyanin, and stellacyanin, in that order. The observation of a nuclear line width of 1.2 MHz is unprecedented in high-resolution NMR in solution. These data are interpreted as a measure of the out-of-plane displacement of the copper ion, which increases on passing from azurin to plastocyanin to stellacyanin. The present approach seems general for the investigation of blue copper proteins.

Basic requirements for a metal-binding site in a protein: The influence of loop shortening on the cupredoxin azurin

“…The g z values of AZAMI-F, AZAMI, and AMI are lower than that for AZ, and the A z values of AZAMI-F and AZAMI are higher than those of AZ and AMI. The paramagnetic 1 H NMR spectrum of AZAMI-F is compared with those of AZAMI (11) and AZ (24) in Fig. 8, which is published as supporting information on the PNAS web site, and has been assigned by using saturationtransfer difference experiments on 1:1 mixtures of oxidized and reduced protein (see Fig.…”

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

“…Again, these correlations are for BCPs with neutral His ligands and H-bonded residues (Az, Pc, Pa, and St), consistent with the results seen with the bond critical point properties. However, when bond critical point (bcp) properties and ring critical point (rcp) properties were used together, the results for all BCPs can be predicted using the following equation: (9) with R 2 = 0.93, 0.97, and 0.94 for ρ(r), G(r), and |V(r)|, respectively. The fitting parameters are listed in Table S37, and the predicted average Cys C β proton NMR hyperfine shifts from eq 9, using bcp and rcp data for ρ(r), G(r), and |V(r)|, are shown in Table 8.…”

“…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.…”

Active Site Comparison of CoII Blue and Green Nitrite Reductases

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14] For the mononuclear type 1 (T1) copper centers found in the cupredoxins [small electron transfer (ET) proteins] studies have shown that a number of isotropically shifted 1 H NMR resonances can be observed and assigned. [1,[8][9][10][11][12][13][14] However, most of these signals are extremely broad due to the relatively slow electronic relaxation rate of the metal and a number of active site resonances cannot be observed. Assignment, and in some cases indirect signal observation (using blind saturation-transfer experiments [8][9][10]12] ), has depended on using the electron self-exchange (ESE) reaction to correlate the shifted signals of the Cu II form with their diamagnetic counterparts in the Cu I cupredoxin.…”