Characterization of a half-apo derivative of peptidylglycine monooxygenase. Insight into the reactivity of each active site copper.
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.
Jaron
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Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, Oregon 97006-8921, USA.
Blackburn
N J
NJ
D000818:Animals; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D016466:CHO Cells; D002248:Carbon Monoxide / Q000737:chemistry; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D006224:Cricetinae; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D009097:Multienzyme Complexes; D009842:Oligopeptides / Q000378:metabolism; D010101:Oxygen Consumption; D013055:Spectrophotometry, Infrared; D013057:Spectrum Analysis; D013379:Substrate Specificity; D014965:X-Rays
D001051:Apoenzymes; D009097:Multienzyme Complexes; D009842:Oligopeptides; D003300:Copper; D002248:Carbon Monoxide; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase