Spectroscopy

Derivatives of CuZnSOD are known with CuII ion bound either to the native copper site or to the native zinc site. The electronic absorption spectra of these derivatives indicate that the ligand environments of the two sites are very different. Copper(1I) is a d 9 transition-metal ion, and its d-d transitions are usually found in the visible and near-IR regions of the spectrum. 53 Copper(1I) complexes with coordinated nitrogen ligands are generally found to have an absorption band between 500 and 700 nm, with an extinction coefficient below 100 M -Icm -I. Bands in the absorption spectra of complexes with geometries that are distorted away from square planar tend to be red-shifted because of a smaller d-d splitting, and to have higher extinction coefficients because of the loss of centrosymmetry. Thus the optical spectrum of CuZnSOD with an absorption band with a maximum at 680 nm (14,700 cm -I; see Figure 5.18A) and an extinction coefficient of 155 M -Icm -I per Cu is consistent with the crystal structural results that indicate that copper(II) is bound to four imidazole nitrogens and a water molecule in a distorted square-pyramidal geometry. Metalsubstituted derivatives with CuII at the native copper site but with COII, CdII, HgII, or Ni II substituted for ZnII at the native zinc site all have a band at 680 nm, suggesting that the substitution of another metal ion for zinc perturbs the copper site very little, despite the proximity of the two metal sites. The absorption spectra of native CuZnSOD and these CuMSOD derivatives also have a shoulder at 417 nm (24,000 cm -I; see Figure 5.18A), which is at lower energy than normal imidazole-to-Cu II charge-transfer transitions, and has been assigned to 

an imidazolate-to-Cu 1I charge transfer, indicating that the imidazolate bridge between CuII and the metal ion in the native zinc site is present, as observed in the crystal structure of CuZnSOD. Derivatives with the zinc site empty, which therefore cannot have an imidazolate bridge, are lacking this 417 nm shoulder. Small but significant changes in the absorption spectrum are seen when the metal ion is removed from the zinc site, e.g., in copper-only SOD (Figure 5.18B). The visible absorption band shifts to 700 nm (14,300 cm -1), presumably due to a change in ligand field strength upon protonation of the bridging imidazolate. In addition, the shoulder at 417 nm has disappeared, again due to the absence of the imidazolate ligand.
 

The spectroscopic properties due to copper in the native zinc site are best observed in the derivative Ag1CuSOD, which has Ag1 in the copper site and Cu II in the zinc site (see Figure 5.18C), since the d 10 Ag1 ion is spectroscopically silent. In this derivative, the dod transition is markedly red-shifted from the visible region of the spectrum into the near-IR, indicating that the ligand environment of CuII in that site is either tetrahedral or five coordinate. The EPR properties of CuII in this derivative are particularly interesting (as discussed below).
 

The derivative with CUll bound at both sites, CuCuSOD, has a visible-near IR spectrum that is nearly a superposition of the spectra of CuZnSOD and Ag1CuSOD (see Figure 5.19), indicating that the geometry of CUll in each of these sites is little affected by the nature of the metal ion in the other site.

EPR spectroscopy has also proven to be particularly valuable in characterizing the metal environments in CuZnSOD and derivatives. The EPR spectrum of native CuZnSOD is shown in Figure 5.20A. The gil resonance is split by the hyperfine coupling between the unpaired electron on CUll and the I = i nuclear spin of copper. The All value, 130 G, is intermediate between the larger All

typical of square-planar CuII complexes with four nitrogen donor ligands and the lower All observed in blue copper proteins (see Chapter 6). The large linewidthseen in the g -l region indicates that the copper ion is in a rhombic (i.e., distorted) environment. Thus, the EPR spectrum is entirely consistent with the distorted square-pyramidal geometry observed in the x-ray structure.
 

Removal of zinc from the native protein to give copper-only SOD results in a perturbed EPR spectrum, with a narrower g1- resonance and a larger All value (142 G) more nearly typical of CUll in an axial N4 environment (Figure 5.20B). Apparently the removal of zinc relaxes some constraints imposed on the geometry of the active-site ligands, allowing the copper to adopt to a geometry closer to its preferred tetragonal arrangement.
 

The EPR spectrum due to Cu II in the native ZnII site in the Ag ICuSOD derivative indicates that Cu II is in a very different environment than when it is in the native copper site (Figure 5.20C). The spectrum is strongly rhombic, with a low value of All (97 G), supporting the conclusion based on the visible spectrum that copper is bound in a tetrahedral or five-coordinate environment. This type of site is unusual either for copper coordination complexes or for copper proteins in general, but does resemble the CUll EPR signal seen when either laccase or cytochrome c oxidase is partially reduced (see Figure 5.21). Partial

reduction disrupts the magnetic coupling between these CuII centers that makes them EPR-silent in the fully oxidized protein.
 

The EPR spectrum of CuCuSOD is very different from that of any of the other copper-containing derivatives (Figure 5.22) because the unpaired spins on

the two copper centers interact and magnetically couple across the imidazolate bridge, resulting in a triplet EPR spectrum. This spectrum is virtually identical with that of model imidazolate-bridged binuclear copper complexes. 

 

Electronic absorption and EPR studies of derivatives of CuZnSOD containing Cu II have provided useful information concerning the nature of the metal binding sites of those derivatives. IH NMR spectra of those derivatives are generally not useful, however, because the relatively slowly relaxing paramagnetic CuII center causes the nearby proton resonances to be extremely broad. This difficulty has been overcome in two derivatives, CuCoSOD and CuNiSOD, in which the fast-relaxing paramagnetic COIl and Ni II centers at the zinc site interact across the imidazolate bridge and increase the relaxation rate of the CuII center, such that well-resolved paramagnetically shifted IH NMR spectra of the region of the proteins near the two paramagnetic metal centers in the protein can be obtained and the resonances assigned. 1l8,119
 

The use of IH NMR to study CuCoSOD derivatives of CuZnSOD in combination with electronic absorption and EPR spectroscopies has enabled investigators to compare active-site structures of a variety of wild-type and mutant CuZnSOD proteins in order to find out if large changes in active-site structure have resulted from replacement of nearby amino-acid residues.