title: Does superoxide channel between the copper centers in peptidylglycine monooxygenase? A new mechanism based on carbon monoxide reactivity.
abstract: Peptidylglycine monooxygenase (PHM) carries out the hydroxylation of the alpha-C atom of glycine-extended propeptides, the first step in the amidation of peptide hormones by the bifunctional enzyme peptidyl-alpha-amidating monooxygenase (PAM). Since PHM is a copper-containing monooxygenase, a study of the interaction between the reduced enzyme and carbon monoxide has been carried out as a probe of the interaction of the Cu(I) sites with O(2). The results show that, in the absence of peptide substrate, reduced PHM binds CO with a stoichiometry of 0.5 CO/Cu(I), indicating that only one of the two copper centers, Cu(B), forms a Cu(I)-carbonyl. FTIR spectroscopy shows a single band in the 2200-1950 cm(-)(1) energy region with nu(CO) = 2093 cm(-)(1) assigned to the intraligand C-O stretch via isotopic labeling with (13)CO. A His242Ala mutant of PHM, which deletes the Cu(B) site by replacing one of its histidine ligands, completely eliminates CO binding. EXAFS spectroscopy is consistent with binding of a single CO ligand with a Cu-C distance of 1.82 +/- 0.03 A. The Cu-S(met) distance increases from 2.23 +/- 0. 02 A in the reduced unliganded enzyme to 2.33 +/- 0.01 A in the carbonylated enzyme, suggesting that the methionine-containing Cu(B) center is the site of CO binding. The binding of the peptide substrate N-Ac-tyr-val-gly perturbs the CO ligand environment, eliciting an IR band at 2062 cm(-)(1) in addition to the 2093 cm(-)(1) band. (13)CO isotopic substitution assigns both frequencies as C-O stretching bands. The CO:Cu binding stoichiometry and peptide/CO FTIR titrations indicate that the 2062 cm(-)(1) band is due to binding of CO at a second site, most likely at the Cu(A) center. This suggests that peptide binding may activate the Cu(A) center toward O(2) binding and reduction to superoxide. As a result of these findings, a new mechanism is proposed involving channeling of superoxide across the 11 A distance between the two copper centers.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, Oregon 97006-8921, USA.
authors.forename: S
authors.initials: S
authors.lastname: Jaron
authors.forename: N J
authors.initials: NJ
authors.lastname: Blackburn
mesh_terms: D000409:Alanine / Q000235:genetics; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002247:Carbon Isotopes; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D003300:Copper / Q000378:metabolism*; D006224:Cricetinae; D006639:Histidine / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes; D017550:Spectroscopy, Fourier Transform Infrared; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013481:Superoxides / Q000737:chemistry / Q000378:metabolism*; D014965:X-Rays
chemical_list: D002247:Carbon Isotopes; D009097:Multienzyme Complexes; D013481:Superoxides; D006639:Histidine; D003300:Copper; D002248:Carbon Monoxide; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D000409:Alanine