Purification and Characterization of Fet3 Protein, a Yeast Homologue of Ceruloplasmin

Silva, Deepika de; Davis‐Kaplan, Sandra; Fergestad, Jennifer; Kaplan, Jerry

doi:10.1074/jbc.272.22.14208

Cited by 115 publications

(104 citation statements)

References 21 publications

(26 reference statements)

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“…These compounds are metalloprotein inhibitors and are thought to competitively bind to the active copper sites where dioxygen binds to the enzyme, thus shutting down the reaction (26). Trapping experiments were repeated in the presence of azide (25 mM) and cyanide (25 mM) to observe its effect on the production of Mn(III) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Evidence for the presence of Mn(III) intermediates in the bacterial oxidation of Mn(II)

Webb

Dick

Bargar

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

292

199

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Bacterial oxidation of Mn(II) to Mn(IV) is believed to drive the oxidative segment of the global biogeochemical Mn cycle and regulates the concentration of dissolved Mn(II) in the oceanic water column, where it is a critical nutrient for planktonic primary productivity. Mn(II) oxidizing activity is expressed by numerous phylogenetically diverse bacteria and fungi, suggesting that it plays a fundamental and ubiquitous role in the environment. This important redox system is believed to be driven by an enzyme or enzyme complex involving a multicopper oxidase, although the biochemical mechanism has never been conclusively demonstrated. Here, we show that Mn(II) oxidation by spores of the marine Bacillus sp. strain SG-1 is a result of two sequential one-step electron transfer processes, both requiring the putative multicopper oxidase, MnxG, in which Mn(III) is a transient intermediate. A kinetic model of the oxidation pathway is presented, which shows that the Mn(II) to Mn(III) step is the rate-limiting step. Thus, oxidation of Mn(II) appears to involve a unique multicopper oxidase system capable of the overall two-electron oxidation of its substrate. This enzyme system may serve as a source for environmental Mn(III), a strong oxidant and competitor for siderophorebound Fe(III) in nutrient-limited environments. That metabolically dormant spores catalyze an important biogeochemical process intimately linked to the C, N, Fe, and S cycles requires us to rethink the role of spores in the environment.kinetics ͉ multicopper oxidase ͉ spores ͉ x-ray absorption near-edge spectroscopy

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Section: Resultsmentioning

confidence: 99%

Evidence for the presence of Mn(III) intermediates in the bacterial oxidation of Mn(II)

Webb

Dick

Bargar

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

292

199

View full text Add to dashboard Cite

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“…Enzyme activities were measured aerobically at room temperature on an SLM Aminco DW-2000 spectrophotometer in the split beam mode. Iron oxidase assays were performed as described by de Silva et al (1997), except that reaction volumes were 1 ml.…”

Section: Methodsmentioning

confidence: 99%

Evidence for a copper-dependent iron transport system in the marine, magnetotactic bacterium strain MV-1

et al. 2004

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Cells of the magnetotactic marine vibrio, strain MV-1, produce magnetite-containing magnetosomes when grown anaerobically or microaerobically. Stable, spontaneous, non-magnetotactic mutants were regularly observed when cells of MV-1 were cultured on solid media incubated under anaerobic or microaerobic conditions. Randomly amplified polymorphic DNA analysis showed that these mutants are not all genetically identical. Cellular iron content of one non-magnetotactic mutant strain, designated MV-1nm1, grown anaerobically, was~20-to 80-fold less than the iron content of wild-type (wt) MV-1 for the same iron concentrations, indicating that MV-1nm1 is deficient in some form of iron uptake. Comparative protein profiles of the two strains showed that MV-1nm1 did not produce several proteins produced by wt MV-1. To understand the potential roles of these proteins in iron transport better, one of these proteins was purified and characterized. This protein, a homodimer with an apparent subunit mass of about 19 kDa, was an iron-regulated, periplasmic protein (p19). Two potential 'copper-handling' motifs (MXM/MX 2 M) are present in the amino acid sequence of p19, and the native protein binds copper in a 1 : 1 ratio. The structural gene for p19, chpA (copper handling protein) and two other putative genes upstream of chpA were cloned and sequenced. These putative genes encode a protein similar to the iron permease, Ftr1, from the yeast Saccharomyces cerevisiae, and a ferredoxin-like protein of unknown function. A periplasmic, copper-containing, iron(II) oxidase was also purified from wt MV-1 and MV-1nm1. This enzyme, like p19, was regulated by media iron concentration and contained four copper atoms per molecule of enzyme. It is hypothesized that ChpA, the iron permease and the iron(II) oxidase might have analogous functions for the three components of the S. cerevisiae copper-dependent high-affinity iron uptake system (Ctr1, Ftr1 and Fet3, respectively), and that strain MV-1 may have a similar iron uptake system. However, iron(II) oxidase purified from both wt MV-1 and MV-1nm1 displayed comparable iron oxidase activities using O 2 as the electron acceptor, indicating that ChpA does not supply the multi-copper iron(II) oxidase with copper.

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“…Other enzymes of the same group are ascorbate oxidase, laccase, and Fet3, a plasma membrane protein of yeasts, recently discovered (13). Among these enzymes, only Cp (14,15) and Fet3 (16) are able to oxidize an inorganic substrate (i.e. Fe(II) ions) in addition to organic substrates such as diamines.…”

Section: Ceruloplasmin (Cp)mentioning

confidence: 99%

Site-directed Mutagenesis of Human Ceruloplasmin

Bielli¹,

Bellenchi²,

Calabrese³

2001

Journal of Biological Chemistry

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A fully active recombinant human ceruloplasmin was obtained, and it was mutated to produce a ceruloplasmin stable to proteolysis. The stable ceruloplasmin was further mutated to perturb the environment of copper at the type 1 copper sites in two different domains. The wild type and the mutated ceruloplasmin were produced in the yeast Pichia pastoris and characterized. The mutations R481A, R701A, and K887A were at the proteolytic sites, did not alter the enzymatic activity, and were all necessary to protect ceruloplasmin from degradation. The mutation L329M was at the tricoordinate type 1 site of the domain 2 and was ineffective to induce modifications of the spectroscopic and catalytic properties of ceruloplasmin, supporting the hypothesis that this site is reduced and locked in a rigid frame. In contrast the mutation C1021S at the type 1 site of domain 6 substantially altered the molecular properties of the protein, leaving a small fraction endowed with oxidase activity. This result, while indicating the importance of this site in stabilizing the overall protein structure, suggests that another type 1 site is competent for dioxygen reduction. During the expression of ceruloplasmin, the yeast maintained a high level of Fet3 that was released from membranes of yeast not harboring the ceruloplasmin gene. This indicates that expression of ceruloplasmin induces a state of iron deficiency in yeast because the ferric iron produced in the medium by its ferroxidase activity is not available for the uptake.

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Purification and Characterization of Fet3 Protein, a Yeast Homologue of Ceruloplasmin

Cited by 115 publications

References 21 publications

Evidence for the presence of Mn(III) intermediates in the bacterial oxidation of Mn(II)

Evidence for the presence of Mn(III) intermediates in the bacterial oxidation of Mn(II)

Evidence for a copper-dependent iron transport system in the marine, magnetotactic bacterium strain MV-1

Site-directed Mutagenesis of Human Ceruloplasmin

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