Aspects of the demetalation and remetalation of ceruloplasmin

Klemens, Frank K.; Severns, John C.; Tamilarasan, R.; McMillin, David R.

doi:10.1016/s0020-1693(96)05213-9

Cited by 2 publications

(1 citation statement)

References 24 publications

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“…Our initial efforts to increase occupancy of copper binding sites in recombinant Hp by treatment with copper sulfate and ascorbate appeared to be unsuccessful (data not shown). In view of the apparent structural similarity of Hp and Cp and the complex procedure required for reconstitution of Cp with copper ( , ), it is likely that reconstitution of Hp with copper will require a similarly complex protocol.…”

Section: Discussionmentioning

confidence: 99%

Recombinant Expression and Functional Characterization of Human Hephaestin: A Multicopper Oxidase with Ferroxidase Activity

2005

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Human hephaestin (Hp) is a transmembrane protein that has been implicated in duodenal iron export. Mutations in the murine hephaestin gene (sla) produce microcytic, hypochromic anemia that is refractory to oral iron therapy. Hp shares approximately 50% sequence identity with the plasma multicopper ferroxidase ceruloplasmin including conservation of residues involved in disulfide bond formation and metal coordination. On the basis of this similarity to ceruloplasmin, human hephaestin may also bind copper and possess ferroxidase activity. To test this hypothesis, human hephaestin cDNA has been cloned by reverse transcription of human duodenal mRNA. Following in vitro mutagenesis to make the encoded polypeptide suitable for expression and purification, the hephaestin cDNA was cloned into the expression vector pNUT and introduced into baby hamster kidney cells. After selection with methotrexate, the baby hamster kidney cells secreted the recombinant human hephaestin into the medium at a level of approximately 2 mg/L. Purification was achieved by a single immunoaffinity chromatography step. As judged by SDS-PAGE, N-terminal sequence analysis, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, the purified hephaestin was homogeneous with a mass of 129600 Da, suggesting a carbohydrate content of 7.7%. Inductively coupled plasma mass spectrometry revealed that recombinant hephaestin contained an average of 3.13 atoms of copper per protein molecule. A visible absorption maximum was observed at 607 nm, consistent with the presence of a Type 1 copper site. By using ferrous ammonium sulfate as a substrate, recombinant hephaestin was shown to have ferroxidase activity with a K(m) of 2.1 microM for Fe(II). Lastly, urea PAGE showed that hephaestin was able to catalyze formation of diferric transferrin from Fe(II) and apotransferrin.

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Section: Discussionmentioning

confidence: 99%

Recombinant Expression and Functional Characterization of Human Hephaestin: A Multicopper Oxidase with Ferroxidase Activity

2005

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Multicopper Oxidase Assembly

Kosman

2017

Encyclopedia of Inorganic and Bioinorganic Chemistry

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The cohort of enzymes designated as multicopper oxidases (MCOs) are expressed by species in all organismal domains. As an enzyme class, these proteins minimally possess a catalytic quartet of copper prosthetic groups that are organized specifically into two, spatially separated (by ∼13 Å) copper centers: (i) a mononuclear copper site—designated as a Type 1 Cu(II)—whose electronic structure and reactivity is dominated by strong S → Cu charge transfer involving an invariant sulfur ligand contributed by a protein cysteine residue; and (ii) a trinuclear copper cluster (TNC) that is linked to the mononuclear site by an invariant –HCH‐sequence motif in which the Cys residue is ligand to the Type 1 Cu and the two His residues are each ligand to one of the Cu atoms in the TNC. This connectivity provides efficient electronic coupling matrix elements for intramolecular electron transfer from the Type 1—as Cu(I)—to the TNC. In MCO turnover, a reducing substrate shuttles electrons to the Type 1 Cu(II) by an outer‐sphere electron transfer pathway; the HCH motif couples this reduced Type 1 Cu(I) to the TNC where O 2 is reduced to 2H 2 O. MCOs are therefore members of a few enzyme classes that reduce dioxygen to water, members that include at the least the terminal heme‐copper oxidases, cytochrome bd oxidases, and type A flavoproteins found in some methanogenic and other bacteria that contain a unique coenzyme F 420 . The Type 1 Cu site in an MCO is homologous to the copper site found in small (∼120 kDa) “blue” copper proteins, for example, azurin and plastocyanin; the Type 1 Cu in an MCO is likely metallated in a process similar to how those proteins characterized by a single cupredoxin domain acquire their prosthetic group. The assembly of the TNC, however, is unique to MCOs and has not been well characterized. This chapter reviews the structural properties of MCO family members and discusses how cell copper trafficking might lead to the activation of these widely distributed members of the copper protein family.

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Aspects of the demetalation and remetalation of ceruloplasmin

Cited by 2 publications

References 24 publications

Recombinant Expression and Functional Characterization of Human Hephaestin: A Multicopper Oxidase with Ferroxidase Activity

Recombinant Expression and Functional Characterization of Human Hephaestin: A Multicopper Oxidase with Ferroxidase Activity

Multicopper Oxidase Assembly

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