Info

Type 1 Type 2 Type 3(2) Type 3(3) Tn nuclear Copper Site

His His His-

Lcu-Lcu-Phe-Lcu-

His His His His

Glv-His-Asp-Phe Glv-His-Ala-Phc Gly-His-Ser-Phe Gly-His-Asp-Phe

Lac 486 Leu-Met-Fet3 481 Phe-Phe-hC'p 1037 Leu-Leu-AO 504 Ala-Phc

His-His His His

Cys Cys Cys Cys

His l!e- Ala-Trp-His: lie- Glu-Trp-i (is Val-Thr-Asp-His Ik- Glu-Pro-

His His His-His-

Val-Ser- Gly- Gly-Leu- Ser-Asn Leu-Leu-GIn-Gly- Leu- Giy-Leu Ile-His- Ala- Gly-Lcu-His-Met-Giv-

Mct. Met

TI/T3 Box

Fouiiii

T] Ligand

Fig. 4. Sequence alignments of the copper-binding motifs in multicopper oxidases. The sequences are for Neurospora crassa laccase (Lac), Cucimus sativus ascorbate oxidase (AO), and two ferroxidases, human ceru-loplasmin (hCp) and Fet3p, from Saccharomyces cerevisiae. The key indicates the ligand assignments to the three copper sites found in these proteins. The sequence numbering is referenced to the ATG that encodes the start codon in the translated mRNA and not to the carboxyl terminal residue of the processed mature protein. Note that all of these are glycoproteins and are targeted to the endoplasmic reticulum. Thus, the nascent polypeptide contains a canonical signal-recognition sequence that is cleaved as part of the proteins' overall processing, which includes the posttranslational insertation of the four active-site copper atoms.

indicates that in Fet3p the NE of His489 (corresponding to His494 and 1045 in Lac and hCp, respectively) would be just at the protein surface and contributing little to the Connelly surface. On the other hand, the He2 hydrogen at His489 would be solvent exposed and H-bonded to water. Further ESEEM analysis was consistent with this inference. The type-1 Cu(II) sites in the three proteins are directly compared in the illustrations shown in Fig. 9.

In addition to the relative degree of solvent exposure represented in these models, the difference in coordination can also be seen. Lac (A) and Fet3p (C) have three-coordinate trigonal type-1 Cu(II) sites, whereas hCp (B) has a four-coordinate distorted tetrahedral structure. Type-1 copper sites in general exhibit this divergence, which can also be seen in the sequence alignments in Fig. 3: AO, like hCp, has the thioether of Met as a fourth ligand. What is interesting is that although this ligand does modulate the properties of type-1 Cu(II) to some extent, other features, such as protein-induced distortion, overall are much stronger determinants of type-1 copper structure and reactivity (2,17).

The ratioed envelope for the type-2 Cu(II) spectrum is best fit with a combination of one equatorial water, one axial water, as well as a distribution of ambient water at a radius of 4-8 Â (Fig. 8E). Together with the Fourier transform shown in Fig. 7B that is attributable to a single equatorially coordinated histidine, the water modulation analysis suggests a structure of the type-2 Cu(II) site in Fet3p that is significantly different from what crystallographic analysis has indicated for the type-2 sites in hCp (7,8) and AO (11,12). This site in these proteins appears to have two equatorial histidine imidazoles, and one equatorial water ligand in an essentially trigonal planar complex. This is illustrated in Fig. 10A. In contrast, Fet3p appears to have only one equatorial histidine imidazole, and one equatorial and one axial water. Inasmuch as the type-2 site in Fet3p has both conserved histidines

Fig. 5. The Connelly surface at the type-1 copper sites in C. cinereus Lac (left) and hCp (right). These surfaces were generated using InsightII software from Molecular Dynamics. The structure files came from the PDB; the accession numbers were 1A65 for Lac, and 1KCW for hCp. The type-1 site in hCp is completely shielded from solvent; H1045 (to the right), which is the closest ligand to the surface, is at least 7 À from it. In contrast, the corresponding His ligand in Lac, His494, is strongly solvent exposed. The shading in the right panel is the result of the NE of this residue and the magenta indicates the adjacent ring carbons. Electron spinecho envelope modulation data indicate that this site in Fet3p is more similar to Lac than to hCp.

Fig. 5. The Connelly surface at the type-1 copper sites in C. cinereus Lac (left) and hCp (right). These surfaces were generated using InsightII software from Molecular Dynamics. The structure files came from the PDB; the accession numbers were 1A65 for Lac, and 1KCW for hCp. The type-1 site in hCp is completely shielded from solvent; H1045 (to the right), which is the closest ligand to the surface, is at least 7 À from it. In contrast, the corresponding His ligand in Lac, His494, is strongly solvent exposed. The shading in the right panel is the result of the NE of this residue and the magenta indicates the adjacent ring carbons. Electron spinecho envelope modulation data indicate that this site in Fet3p is more similar to Lac than to hCp.

(His81 and His416), it is possible that the second of these is an axial ligand at this site and therefore does not contribute to the modulation pattern. This model of the type-2 site is presented in Fig. 10B. A model for all three copper sites in Fet3p and there putative spatial relationship was given in Fig. 3.

6. EXAFS ANALYSIS OF FET3P COPPER-SITE STRUCTURE: THE BINUCLEAR TYPE-3 SITE

One recombinant Fet3p mutant has been particularly informative about the structure of the type-3 binuclear cluster in them. This is the T1D/T2D double mutant that contains only this type-3 site (5). EXAFS (extended X-ray absorption fine structure) analysis of this protein contains contributions from electron ejection and scattering from only the type-3 copper atoms and thus provides direct structural information about this cluster. The K-edge XAS spectrum for this mutant in its oxidized and reduced states is shown in Fig. 11. The oxidized sample has a nearly featureless edge with a midpoint energy of 8990 eV typical of tetragonally distorted type-2 Cu(II) centers (i.e., ones with predominantly histidine imidazole coordination). The reduced type-3 cluster exhibited a pronounced shoulder at 8984 eV just below the midpoint energy of 8987 eV. The shoulder is characteristic of a

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