Metal-replacement studies of blue copper proteins

Morris, Margaret C.; Hauenstein, Bennett L.; McMillin, David R.

doi:10.1016/0167-4838(83)90397-7

Cited by 9 publications

(4 citation statements)

References 17 publications

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“…Furthermore, a systematic analysis of the effect of copper depletion on the sensitivity of AO to thermal and chemical denaturation concluded that copper affects the stability but not the protein conformation (50). Similar observations have been made with laccases, which almost completely regains the activity, copper content, and spectroscopic properties of the native protein upon treatment of the apoprotein with cuprous ion (51,52). In contrast, apo-hCp, fully depleted from its copper atoms as studied here, is unable to recover its copper complement and its enzymatic activity (8).…”

Section: Discussionmentioning

confidence: 58%

A Key Structural Role for Active Site Type 3 Copper Ions in Human Ceruloplasmin

Vachette

Dainese

Vasyliev

et al. 2002

Journal of Biological Chemistry

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Human ceruloplasmin is a copper containing serum glycoprotein with multiple functions. The crystal structure shows that its six domains are arranged in three pairs with a pseudo-ternary axis. Both the holo and apo forms of human ceruloplasmin were studied by size exclusion chromatography and small angle x-ray scattering in solution. The experimental curve of the holo form displays conspicuous differences with the scattering pattern calculated from the crystal structure. Once the carbohydrate chains and flexible loops not visible in the crystal are accounted for, remaining discrepancies suggest that the central pair of domains may move as a whole with respect to the rest of the molecule. The quasisymmetrical crystal structure therefore appears to be stabilized by crystal packing forces. Upon copper removal, the scattering pattern of human ceruloplasmin exhibits very large differences with that of the holoprotein, which are interpreted in terms of essentially preserved domains freely moving in solution around flexible linkers and exploring an ensemble of open conformations. This model, which is supported by the analysis of domain interfaces, provides a structural explanation for the differences in copper reincorporation into the apoprotein and activity recovery between human ceruloplasmin and two other multicopper oxidases, ascorbate oxidase and laccase. Our results demonstrate that, beyond catalytic activity, the three-copper cluster at the N-terminal-C-terminal interface plays a crucial role in the structural stability of human ceruloplasmin.

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Section: Discussionmentioning

confidence: 58%

A Key Structural Role for Active Site Type 3 Copper Ions in Human Ceruloplasmin

Vachette

Dainese

Vasyliev

et al. 2002

Journal of Biological Chemistry

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“…The completely demetallated proteins were prepared by dialysis against KCN according to previously published methods (Morpurgo et al, 1972;McMillin et al, 1974;Morris et al, 1983). The apo-azurin preparation (McMillin et al, 1974) was modified such that the 50 mM-cyanide solution was in 0.1 M-imidazolium acetate with a final pH of 7 rather than the prescribed 25 M-Tris buffer at pH 8.…”

Section: Methodsmentioning

confidence: 99%

Cu(I) analysis of blue copper proteins

Hanna

Tamilarasan

McMillin

1988

Biochemical Journal

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A simple colorimetric test for the Cu(I) content in blue copper proteins is described. The procedure is based on the formation of a complex between Cu(I) and 2,2'-biquinoline in an acetic acid medium. Analyses of spinach plastocyanin, Pseudomonas aeruginosa azurin and Rhus vernicifera stellacyanin show that the cysteine residue in the type 1 site does not induce Cu(II) reduction under our conditions. There is evidence in laccase samples for the presence of an endogenous reductant that can reduce 0.14 +/- 0.04 mol of Cu(II)/mol of protein; however, the addition of EDTA eliminates the interference. The analysis shows that 25 +/- 2% of the type 3 copper ions are in the reduced form in the resting enzyme and that 80 +/- 15% of the type 3 copper ions are reduced in preparations of type-2-depleted laccase. There is growing interest in the development of chemically modified forms of laccase, and our method should be very useful for establishing the valence state of the metal centres in the various derivatives.

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“…Stated differently the type 1 and type 3 Cu of fungal laccase appear to be much more difficult to remove than the type 2 Cu. This provides another contrast with the tree enzyme, where copper can easily be removed from all three binding sites by dialysis against cyanide at pH 7 [22]. Electronic absorption and c.d.…”

Section: Failed Reconstitution Of Apolaccasementioning

confidence: 98%

“…The fact that the type 1 Cu site of the apoenzyme is not restored is interesting, since this site is readily reconstituted in a variety of other blue Cu proteins [21], including tree laccase [22]. This could imply that the fungal protein adopts a quite different structure when Cu is removed and that this process is not readily reversed.…”

Section: Failed Reconstitution Of Apolaccasementioning

confidence: 99%

Type 2-depleted fungal laccase

Hanna

McMillin

Pasenkiewicz-Gierula

et al. 1988

Biochemical Journal

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Although copper is quantitatively removed from fungal laccase (Polyporus versicolor) by extended dialysis against high concentrations of cyanide, we have been unable to reconstitute the protein by addition of Cu(I) ions. However, two new methods for reversibly removing the type 2 Cu centre have been developed. The visible absorption at 610 nm, which is attributable to type 1 Cu, is unaffected by the procedure, but the absorbance of the type 3 Cu at 330 nm is decreased by 60 +/- 10%. The decrease is due, at least in part, to partial reduction of the binuclear type 3 centre, although there may be some change in the molar absorptivity of the oxidized chromophore as well. The change in the c.d. spectrum that occurs at approx. 350 nm may be explained in the same way, but it may also reflect the loss of a signal due to the type 2 Cu. Upon removal of the type 2 Cu an absorbance increase appears at approx. 435 nm, and it is assigned to the semi-reduced form of the type 3 pair. In the e.p.r. spectrum of the type 2-depleted enzyme the type 1 Cu signal exhibits well-resolved ligand hyperfine splitting, which can be simulated on the basis of contributions from two N and two H nuclei (AH congruent to AN congruent to 25 MHz). The H atoms are assumed to be attached to the beta-carbon of the covalently bonded cysteine ligand. A signal from a semi-reduced form(s) of the type 3 site can also be resolved in the spectrum of the type 2-depleted enzyme, and on the basis of the second integral of the e.p.r. spectrum 40% of the type 3 pairs are believed to be in a partially reduced state. The semi-reduced type 3 site is remarkably stable and is not readily oxidized by H2O2 or IrCl6(2-) or reduced by Fe(CN)6(4-). Intramolecular electron transfer is apparently quite slow in at least some forms of the type 2-depleted enzyme, and this may explain why the activity is at best 5% of that of the native enzyme. Full activity returns when type 2 copper is restored.

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Metal-replacement studies of blue copper proteins

Cited by 9 publications

References 17 publications

A Key Structural Role for Active Site Type 3 Copper Ions in Human Ceruloplasmin

A Key Structural Role for Active Site Type 3 Copper Ions in Human Ceruloplasmin

Cu(I) analysis of blue copper proteins

Type 2-depleted fungal laccase

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