The role of cyanide in the removal of type 2 copper from laccase

Eggleston, Michèle K.; Pecoraro, Catherine; McMillin, David R.

doi:10.1016/0003-9861(95)90010-1

Cited by 5 publications

(3 citation statements)

References 18 publications

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“…Also, there were only small differences in the g and A values (Figure 1 and the text), consistent with little change in the electronic structure of the remaining Cu-(II) site in these two single-mutant proteins. This result was similar to what has been observed in the T1Hg (38,43,44) and T2D Lac (39) and T2D AO (40) species. Also, the type 3 binuclear Cu(II) cluster in the T1D/T2D Fet3 protein exhibited a comparable near-UV absorption, as well as absorbance at ∼720 nm.…”

Section: Discussionsupporting

confidence: 91%

“…Single "deletion mutants" have been generated previously at the type 1 and type 2 copper sites in multinuclear copper oxidases by chemical means. Thus, metal substitution at the type 1 copper, e.g., mercury, to yield T1Hg Lac (38), or metal depletion by chelation in the presence of a reducing agent as in the preparation of T2D forms of Lac (39) and AO (40), has been used to generate protein forms that lacked one of the copper sites. Here, we report for the first time the use of recombinant DNA technology in generating true site-specific deletion mutants at the copper sites in a multinuclear copper oxidase.…”

Section: Discussionmentioning

confidence: 99%

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Spectroscopic Analysis of the Trinuclear Cluster in the Fet3 Protein from Yeast, a Multinuclear Copper Oxidase

et al. 2000

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The Fet3 protein (Fet3p) is a multinuclear copper oxidase essential for high-affinity iron uptake in yeast. Fet3p contains one type 1, one type 2, and a strongly antiferromagnetically coupled binuclear Cu(II)-Cu(II) type 3 copper. The type 2 and type 3 sites constitute a structurally distinct trinuclear cluster at which dioxygen is reduced to water. In Fet3p, as in ceruloplasmin, Fe(II) is oxidized to Fe(III) at the type 1 copper; this is the ferroxidase reaction that is fundamental to the physiologic function of these two enzymes. Using site-directed mutagenesis, we have generated type 1-depleted (T1D), type 2-depleted (T2D), and T1D/T2D mutants. None were active in the essential ferroxidase reaction catalyzed by Fet3p. However, the spectroscopic signatures of the remaining Cu(II) sites in any one of the three mutants were indistinguishable from those exhibited by the wild type. Although the native protein and the T1D mutant were isolated in the completely oxidized Cu(II) form, the T2D and T1D/T2D mutants were found to be completely reduced. This result is consistent with the essential role of the type 2 copper in dioxygen turnover, and with the suggestions that cuprous ion is the valence state of intracellular copper. Although stable to dioxygen, the Cu(I) sites in both proteins were readily oxidized by hydrogen peroxide. The double mutant was extensively analyzed by X-ray absorption spectroscopy. Edge and near-edge features clearly distinguished the oxidized from the reduced form of the binuclear cluster. EXAFS was strongly consistent with the expected coordination of each type 3 copper by three histidine imidazoles. Also, copper scattering was observed in the oxidized cluster along with scattering from a ligand corresponding to a bridging oxygen. The data derived from the reduced cluster indicated that the bridge was absent in this redox state. In the reduced form of the double mutant, an N/O ligand was apparent that was not seen in the reduced form of the T1D protein. This ligand in T1D/T2D could be either the remaining type 2 copper imidazole ligand (from His416) or a water molecule that could be stabilized at the type 3 cluster by H-bonding to this side chain. If present in the native protein, this H(2)O could provide acid catalysis of dioxygen reduction at the reduced trinuclear center.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Spectroscopic Analysis of the Trinuclear Cluster in the Fet3 Protein from Yeast, a Multinuclear Copper Oxidase

et al. 2000

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“…Several studies show selective depletion of type 2 copper ion from various types of multicopper oxidases [14][15][16][17][18][19][20][21][22]. Different reagents have been described in the literature as potential chelators for selectively removing the type 2 copper ion [19,21,[23][24][25][26][27]. The possible interaction between the type 1 and type 3 sites has also been studied.…”

Section: Introductionmentioning

confidence: 99%

The reversible depletion and reconstitution of a copper ion in Coprinus cinereus laccase followed by spectroscopic techniques

Bukh

Bjerrum

2010

Journal of Inorganic Biochemistry

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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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The role of cyanide in the removal of type 2 copper from laccase

Cited by 5 publications

References 18 publications

Spectroscopic Analysis of the Trinuclear Cluster in the Fet3 Protein from Yeast, a Multinuclear Copper Oxidase

Spectroscopic Analysis of the Trinuclear Cluster in the Fet3 Protein from Yeast, a Multinuclear Copper Oxidase

The reversible depletion and reconstitution of a copper ion in Coprinus cinereus laccase followed by spectroscopic techniques

Multicopper Oxidase Assembly

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