Characterization of a half-apo derivative of peptidylglycine monooxygenase. Insight into the reactivity of each active site copper

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)6867-6875
Number of pages9
JournalBiochemistry
Volume40
Issue number23
DOIs
StatePublished - Jun 12 2001

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Carbon Monoxide
Copper
Catalytic Domain
Derivatives
Enzymes
Peptides
Proteins
Substrates
Histidine
Methionine
peptidylglycine monooxygenase
Binding Sites
Molecules
Experiments

ASJC Scopus subject areas

  • Biochemistry

Cite this

Characterization of a half-apo derivative of peptidylglycine monooxygenase. Insight into the reactivity of each active site copper. / Jaron, S.; Blackburn, Ninian.

In: Biochemistry, Vol. 40, No. 23, 12.06.2001, p. 6867-6875.

Research output: Contribution to journalArticle

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N2 - 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.

AB - 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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