Removal of copper from <i>Octopus vulgaris</i> haemocyanin. Preparation of the half-apo and apo derivatives

Beltramini, Mariano; Ricchelli, Fernanda; Piazzesi, A. M.; Barel, Alessandra; Salvato, Benedetto

doi:10.1042/bj2210911

Cited by 10 publications

(1 citation statement)

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“…Cyanide is known to compete with oxygen for the Cu(I) binding sites in proteins such as DβM (22,32,33) and hemocyanin (34,35) due to its propensity to both coordinate and remove copper from the proteins. Results from the cyanide dialysis of native PHM show that demetalated derivatives can likewise be formed in this enzyme.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Half-Apo Derivative of Peptidylglycine Monooxygenase. Insight into the Reactivity of Each Active Site Copper

Jaron

Blackburn

2001

Biochemistry

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A derivative of peptidylglycine monooxygenase which lacks the CuH center has been prepared and characterized. This form of the enzyme is termed the half-apo protein. Copper-to-protein stoichiometric measurements establish that the protein binds only one of the two copper centers (CuM and CuH) found in the native enzyme. Confirmation that the methionine-containing CuM has been retained has been obtained from EXAFS experiments which show that the characteristic signature of the Cu-S(Met) interaction is preserved. The half-apo derivative binds 1 equiv of CO per copper with an IR frequency of 2092 cm(-1), and this monocarbonyl also displays the Cu-S(Met) interaction in its EXAFS spectrum. These results allow unambiguous assignment of the 2092 cm(-1) band as a CuM-CO species. Binding of CO in the presence of peptide substrate was also investigated. In the native enzyme, substrate induced binding of a second CO molecule with an IR frequency of 2062 cm(-1), tentatively assigned to a CO complex of the histidine-containing CuH site. Unexpectedly, this reactivity is also observed in the half-apo derivative, although the intensity distribution of the CO stretches now indicates that the copper has been partially transferred to a second site, believed to be CuH. The implications of this observation are discussed in terms of a possible additional peptide binding site close to the CuH center.

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