Proton NMR Spectroscopy as a Probe of Dinuclear Copper(II) Active Sites in Metalloproteins. Characterization of the Hyperactive Copper(II)-Substituted Aminopeptidase from Aeromonas proteolytica

Abstract

Proton NMR spectra of the hyperactive Cu(II)-substituted aminopeptidase from Aeromonas proteolytica (AAP) were recorded in both H2O and D2O buffered solution at pH 6.7. Several remarkably sharp, well resolved hyperfine shifted 1H NMR signals were observed in the 70 to −20 ppm chemical shift range. That hyperfine shifted signals were observed is due to spin-coupling of the two Cu(II) ions. Comparison of the spectra recorded in H2O and D2O buffered solutions indicated that the signals at 44.6, 43.3, and 17.7 ppm were solvent exchangeable. The two most strongly downfield shifted signals were assigned to imidazole N−H protons of the two coordinated histidine residues, while the remaining exchangeable signal was assigned to a peptidyl N−H proton that is in close proximity to the dicopper(II) center. One-dimensional NOE studies at pH 6.7 revealed two Y−CH2−CH< moieties that were assigned to coordinated aspartic acid and histidine residues. In addition, a Y−CH2−CH2−CH< moiety was also identified and was assigned to the coordinated glutamic acid residue, Glu152. All of the hyperfine shifted signals for [CuCu(AAP)] sharpened and shifted toward the diamagnetic region as the temperature was increased following Curie behavior. Fits of these data and those of a series of magnetically diverse μ-phenoxo and μ-alkoxo dicopper(II) model complexes to the population distribution of the ground and first excited states, provided information on the magnetic properties of dicopper(II) clusters. These fits indicated that the two Cu(II) ions in AAP are ferromagnetically coupled with a 2J value of 50 + 40 cm-1. These data provide the first structural information regarding the hyperactive [CuCu(AAP)] enzyme and are discussed in terms of the previously proposed mechanism of action for AAP.

Structure and Mechanism of Copper- and Nickel-Substituted Analogues of Metallo-β-lactamase L1

“…In addition, even strongly antiferromagnetically-coupled Cu(II)-Cu(II), e.g. 50 cm −1 (33), would have substantially populated excited states at ambient temperatures, and NMR signals would be expected. The available data suggest, then, that the low-affinity Cu site is not the Zn 2 site.…”

Towards a Protocol for Solution Structure Determination of Copper(II) Proteins: the Case of CuIIZnII Superoxide Dismutase

“…Chemical shift information A comparison of the 1 H, 15 N HSQC spectra of oxidized and reduced SOD (Figure 1) indicates that 52 signals are missing in the oxidized, paramagnetic form of the enzyme. The corresponding residues are located within a sphere around the copper ion of about an 11 radius, where there is no chemical shift information.…”

“…Type 3 copper proteins have a pair of magnetically coupled copper(II) ions, and this has A C H T U N G T R E N N U N G favorable consequences on the observability of the NMR signals. [14][15][16] In type 2 Cu II proteins, proton NMR signals are broadened beyond detection within a sphere of about 10-11 from the metal ion. [1] Thus, structural information cannot be obtained within this region.…”

Metallopeptides — from Drug Discovery to Catalysis

“…We have utilized paramagnetic metal ions extensively as NMR probes for structural and mechanistic studies of a variety of metal complexes, 41,42 metalloproteins, [43][44][45][46][47][48][49][50][51][52][53] and metallodrugs. 4,33,[54][55][56] Owing to the paramagnetism, NMR signals of protons in close proximity of the metal are hyperfine-shifted outside the normal spectral window of less than 15 ppm in a large range which may reach more than 100 ppm in many cases.…”