The multicopper oxidases are a family of enzymes that couple the reduction of O(2) to H(2)O with the oxidation of a range of substrates. Saccharomyces cerevisiae Fet3p and human ceruloplasmin (hCp) are members of this family that exhibit ferroxidase activity. Their high specificity for Fe(II) has been attributed to the existence of a binding site for iron. In this study, mutations at the E185 and Y354 residues, which are putative ligands for iron in Fet3p, have been generated and characterized. The effects of these mutations on the electronic structure of the T1 Cu site have been assessed, and the reactivities of this site toward 1,4-hydroquinone (a weak binding substrate) and Fe(II) have been evaluated and interpreted in terms of the semiclassical Marcus theory for electron transfer. The electronic and geometric structure of the Fe(II) substrate bound to Fet3p and hCp has been studied for the first time, using variable-temperature variable field magnetic circular dichroism (VTVH MCD) spectroscopy. The iron binding sites in Fet3p and hCp appear to be very similar in nature, and their contributions to the ferroxidase activity of these proteins have been analyzed. It is found that these iron binding sites play a major role in tuning the reduction potential of iron to provide a large driving force for the ferroxidase reaction, while still supporting the delivery of the Fe(III) product to the acceptor protein. Finally, the analysis of possible electron-transfer (ET) pathways from the protein-bound Fe(II) to the T1 Cu site indicates that the E185 residue not only plays a role in iron binding, but also provides the dominant ET pathway to the T1 Cu site.

• PMID: 15161286
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Comparative Study | Journal Article | Research Support, U.S. Gov't, P.H.S.

Authors

Quintanar L, Gebhard M, Wang TP, Kosman DJ, Solomon EI

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