D

500 1000 1500 2000 2500 (t, ns)

Fig. 8. Simulated and experimental two-pulse 2H2O (solvent water) ESEEM spectra. Theoretical ESEEM spectra for equatorial, axial, and ambient water are calculated as indicated. These can be compared to the experimental envelopes for the Fet3p type-1 and type-2 Cu(II) sites (solid lines) and the simulations for these envelopes assuming only ambient water for the type-1 copper and a combination of one equatorial, one axial, and ambient water (dotted lines) for the type-2 copper. (From ref. 14.)

500 1000 1500 2000 2500 (t, ns)

Fig. 8. Simulated and experimental two-pulse 2H2O (solvent water) ESEEM spectra. Theoretical ESEEM spectra for equatorial, axial, and ambient water are calculated as indicated. These can be compared to the experimental envelopes for the Fet3p type-1 and type-2 Cu(II) sites (solid lines) and the simulations for these envelopes assuming only ambient water for the type-1 copper and a combination of one equatorial, one axial, and ambient water (dotted lines) for the type-2 copper. (From ref. 14.)

have proposed that this patch contains two ligand arrays essential to the ferroxidase reaction (8). First, they interpreted a region of electron density adjacent to the type-1 Cu(II) as a "labile" copper-binding site that includes residues E291, E954, H959, and D1044. (This numbering is referenced to the mRNA sequence that includes the signal sequence cleaved during processing. This notation has been chosen to facilitate genome-based alignments.) They postulate that Fe2+ binds at this site and is oxidized in an outer-sphere electron transfer to the type-1 Cu(II). His959 and/or D1044 could provide the path for this process because both are in contact with the two histidine imidazoles that are ligands to the type-1 copper. Immediately adjacent to and on the solvent side of this "labile" site is a region in which additional electron density is observed when the crystals are soaked in Fe3+. The authors refer to this as the Fe3+ holding site for the ferric ion product of the ferroxidase reaction. The ligands at this putative Fe3+ site are not well delineated. However, D249, E950, E951, and E976 are in the vicinity of this electron density. Also, the electron density resulting from E954 shifts away from the "labile" site toward this "holding" site, suggesting that this side chain could play a role in the channeling of the Fe3+ from one site to the other. As appealing as this model is, it is based on fairly limited structural data. Systematic structure-function studies have yet to be done to test this model (illustrated in Fig. 13).

Fet3p most certainly does not have the trimeric structure that Cp has and thus cannot have the ligand arrays that Lindley and co-workers propose for the "labile" and "holding" sites. In both sites, the ligands come from at least two of the six domains. There is nothing in the Fet3p sequence to indicate that Fet3p has a tertiary fold of this nature. On the other hand, Fet3p most reasonably has one or more ligand arrays that play a similar function. What the crystallographic data on hCp and their interpretation suggest is that these arrays would be composed of similar

7.2. Fet3p

Fig. 9. Model of the type-1 Cu(II) structures in Lac, hCp, and Fet3p. The solvent accessibility is indicated by the shading. As shown in Fig. 5, His494 in Lac is strongly solvent exposed, whereas in hCp, His1045 is fully buried within the protein. ESEEM analysis (Fig. 8) suggests that this residue in Fet3p, His489, is at or near the surface of the protein, more similar to Lac than to hCp.

Fig. 9. Model of the type-1 Cu(II) structures in Lac, hCp, and Fet3p. The solvent accessibility is indicated by the shading. As shown in Fig. 5, His494 in Lac is strongly solvent exposed, whereas in hCp, His1045 is fully buried within the protein. ESEEM analysis (Fig. 8) suggests that this residue in Fet3p, His489, is at or near the surface of the protein, more similar to Lac than to hCp.

amino acid side-chain types, specifically D or E and H residues. Askwith and Kaplan based mutagenesis studies on the very reasonable assumption that such residues would provide this type of metal-ion coordination (18). Based a homology modeling study carried out by Murphy et al. (20) and sequence alignments among the putative multicopper oxidases found in a variety of yeast genomes (shown in Fig. 14), they prepared several Fet3p mutants. These were tested in vivo for their ability to support iron uptake and in vitro for their ferroxidase activity as visualized by a histochemical enzyme stain of membrane extracts fractionated in polyacrylamide gels. Among these mutants were E227A, D228A, and E230A. These are found in "ferroxidase" Box 3

Fig. 10. Alternative type-2 Cu(II) coordination. (A) The structure of the type-2 site in AO and hCp is represented in which the two His ligands define a single, presumably equatorial plane. An equatorial H2O is indicated by the fact that anions, including peroxide, the two-electron oxygen reduction intermediate, does bind equatorially in AO (11). (B) The type-2 Cu(II) coordination proposed for Fet3p. ESEEM indicates that this site has only one equatorial His, and one equatorial and one axial water. The other His at this site may be axial because it does not contribute to the N modulation in the ESEEM pattern (14).

Fig. 10. Alternative type-2 Cu(II) coordination. (A) The structure of the type-2 site in AO and hCp is represented in which the two His ligands define a single, presumably equatorial plane. An equatorial H2O is indicated by the fact that anions, including peroxide, the two-electron oxygen reduction intermediate, does bind equatorially in AO (11). (B) The type-2 Cu(II) coordination proposed for Fet3p. ESEEM indicates that this site has only one equatorial His, and one equatorial and one axial water. The other His at this site may be axial because it does not contribute to the N modulation in the ESEEM pattern (14).

8960 8980 9000 9020 Energy (eV)

Fig. 11. XAS of the binuclear type-3 copper site in a Fet3p double mutant. The T1D/T2D Fet3p sample was 0.5 mM in pH = 6.0 MES buffer at 11-14 K. The data were collected at the Stanford Synchrotron Radiation Laboratory (5). The XAS for the oxidized and reduced type-3 sites are indicated.

(Fig. 14), and except for the latter residue in Fiolp, they are fully conserved in all the yeast "ferroxidases" identified by sequence analysis to date. Based on the structural model, all were predicted to play a role in either the binding of Fe2+, or the electron-transfer reaction, or both (19,20). In fact, none of these mutants constructed by Askwith and Kaplan exhibited any significant loss of ferroxidase activity in this qualitative histochemical assay, and all supported a normal rate of iron uptake. Possibly, a more rigorous kinetic analysis would reveal some deficit in enzymic activity in one or more of these mutant proteins. However, in light of the normal physiologic activity of these several-mutant proteins, it is highly unlikely that any one of these residues plays a critical role in the structure and function of Fet3p.

Fig. 12. Model for the coordination changes at the binuclear copper site in Fet3p upon reduction. This model is based on the fitting of the EXAFS data from the oxidized and reduced forms of T1D and T1D/T2D Fet3p. This fitting indicated three significant structural differences upon cluster reduction: (1) loss of the bridging oxygen ligand; (2) separation of the two copper atoms; (3) appearance of a nonbridging O/N ligand with a relatively long bond to one of the copper atoms. Also pictured in the model for the reduced state is the dioxygen liganded to Cu(2), as has been proposed based on a variety of spectral and kinetic data. The model here suggests that the proposed water at Cu(3) that results from protonation of the bridging (-OH) upon reduction could serve as an acid catalyst of the reduction of the bound O2. (From ref. J.)

Fig. 12. Model for the coordination changes at the binuclear copper site in Fet3p upon reduction. This model is based on the fitting of the EXAFS data from the oxidized and reduced forms of T1D and T1D/T2D Fet3p. This fitting indicated three significant structural differences upon cluster reduction: (1) loss of the bridging oxygen ligand; (2) separation of the two copper atoms; (3) appearance of a nonbridging O/N ligand with a relatively long bond to one of the copper atoms. Also pictured in the model for the reduced state is the dioxygen liganded to Cu(2), as has been proposed based on a variety of spectral and kinetic data. The model here suggests that the proposed water at Cu(3) that results from protonation of the bridging (-OH) upon reduction could serve as an acid catalyst of the reduction of the bound O2. (From ref. J.)

Fig. 13. Model of the iron-binding sites in hCp. Fe2+ oxidation is proposed to occur with the metal bound at a site adjacent to the type-1 copper. Crystallographic data suggest that E291, E954, H959, and D1044 are ligands to the iron at this site (8). The Fe3+ produced is suggested to then migrate to a "holding site" that is closer to the protein surface. The ligation of this site is not resolved crystallographically; the residues indicated are potential ligands.

Fig. 13. Model of the iron-binding sites in hCp. Fe2+ oxidation is proposed to occur with the metal bound at a site adjacent to the type-1 copper. Crystallographic data suggest that E291, E954, H959, and D1044 are ligands to the iron at this site (8). The Fe3+ produced is suggested to then migrate to a "holding site" that is closer to the protein surface. The ligation of this site is not resolved crystallographically; the residues indicated are potential ligands.

Fig. 14. Alignment of encoded sequences of Fet3p (gi6323703) and Fet5p (gi6321067) from Saccharomy-ces cerevisiae, and Fet homolog from Candida albicans (gi1684656), and Fio1p (gi1067210) from Schizosaccharomyces pombe. The copperligands are indicated by • . The four homology elements common to the fungal "ferroxidases" are noted as "boxes." Within two of these boxes, specific residues have been mutated. These are indicated by an asterisk (*). The arrow below the Leu residue in the type-1 site of these fungal proteins shows that, as a group, this site is trigonal in this class of multicopper oxidases. The bar above residues 560-579 in Fet3p indicates the predicted carboxyl-terminal membrane-spanning element in this protein; the alignment shows that this element is likely conserved in Fet5p (a known type-1 membrane protein) and the Fet homolog from C. albicans but is most likely absent in Fio1p. These sequences and others noted in the text can be retrieved from the website maintained by the National Center of Biotechnology Information (NCBI) using the accession numbers given (www.ncbi.nlm.nih.gov).

Fig. 14. Alignment of encoded sequences of Fet3p (gi6323703) and Fet5p (gi6321067) from Saccharomy-ces cerevisiae, and Fet homolog from Candida albicans (gi1684656), and Fio1p (gi1067210) from Schizosaccharomyces pombe. The copperligands are indicated by • . The four homology elements common to the fungal "ferroxidases" are noted as "boxes." Within two of these boxes, specific residues have been mutated. These are indicated by an asterisk (*). The arrow below the Leu residue in the type-1 site of these fungal proteins shows that, as a group, this site is trigonal in this class of multicopper oxidases. The bar above residues 560-579 in Fet3p indicates the predicted carboxyl-terminal membrane-spanning element in this protein; the alignment shows that this element is likely conserved in Fet5p (a known type-1 membrane protein) and the Fet homolog from C. albicans but is most likely absent in Fio1p. These sequences and others noted in the text can be retrieved from the website maintained by the National Center of Biotechnology Information (NCBI) using the accession numbers given (www.ncbi.nlm.nih.gov).

One of the two systems that has been developed to produce soluble, recombinant Fet3p involves expression of FET3 in insect cells using the standard Baculovirus system (21). The protein produced localizes to the cell membrane from which it can be released in soluble form by mild trypsin digestion. The protein released is in a copper-free apo form and must be incubated with copper in vitro to attain enzymatic activity. In this system, two mutants were constructed based on a model of Fet3p built on the structure of ascorbate oxidase (19,20). In this model, residues E185 and Y354 were located adjacent to the type-1 site and therefore were proposed to be involved in iron binding. The inclusion of Y354 in this set is somewhat surprising inasmuch as Y would preferentially bind Fe3+, not Fe2+. On the other hand, both residues are fully conserved among the yeast ferroxidase homologs (see Fig. 14, Box 2 and Box 4). In any event, only the E185A mutant exhibited strikingly different kinetics with Fe2+ as substrate. For this mutant, Vmax was reduced 2.5-fold while Km for Fe2+ was increased 6-fold. The result of these two changes was a 16-fold decrease in Vmax/Km for Fe2+ turnover. Steady-state results like these are ambiguous with respect to mechanism because these kinetic constants can reflect the contributions of several discreet steps in the overall enzymic reaction. Among these are the intermolecular and intramolecular electron-transfer steps, the electron transfers to O2, and the release of H2O. Therefore, it is not possible to conclude from this type of experiment that a particular amino acid residue is playing a specific role in only one of these steps.

Nonetheless, mutation at E185 expressed in the Baculovirus system did alter the ferroxidase activity of Fet3p, indicating that this residue could be a candidate for one of the ligands involved in iron binding and/or electron transfer into the type-1 Cu(II). These initial data indicated that the role of E185 needed to be thoroughly delineated as noted, particularly because it is found within one of the highly conserved motifs that appear to distinguish the yeast ferroxidases from those found in other eukaryotes (e.g., flies and mammals). Therefore, it could have played a specific role in the ferroxidase reaction of Fet3p and there may not be a functionally homologous residue in hCp.

On the other hand, one can reasonably question the choice of structural template in either of these modeling studies. As noted, Fet3p and hCp have little in common except the copper liganding motifs characteristicsof all multicopper oxidases (Fig. 4). Although more similar to Fet3p, AO, used by Buonaccorsi di Patti et al. in their modeling studies, also has a domain structure that sequence alignments indicate are not likely to be found in Fet3p. The most similar to Fet3p of those multicopper oxidases for which structural coordinates have been published is C. cinereus laccase. In a structure of Fet3p built in silico based on the Lac structure, E185 and Y354 are both over 20 A away from the type-1 copper site (Kosman, unpublished). The location of E185 in Fet3p based on this model is shown in Fig. 15A. Of course, this model is no more likely to be correct than either of the other two. Clearly, truly useful knowledge about the structure of Fet3p with regard to residues with the potential to contribute to substrate specificity will come only from the crystallographically determined structure itself.

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