title: Direct infrared detection of the covalently ring linked His-Tyr structure in the active site of the heme-copper oxidases.
abstract: Infrared spectroscopy, isotopic labeling ([(15)N(delta,epsilon)]histidine and ring-deuterated tyrosine), synthetic model studies, and normal mode calculations are employed to search for the spectroscopic signatures of the unique, covalently linked (His N(epsilon)-C(epsilon) Tyr) biring structure in the heme-copper oxidases. The specific enzyme examined is the cytochrome bo(3) quinol oxidase of E. coli. Infrared features of histidine and tyrosine are identified in the frequency regions of imidazole and phenol ring stretching modes (1350-1650 cm(-1)) and C-H and N-H stretching modes as well as overtones and combinations (>3000 cm(-1)). Two of these, at ca. 1480 and 1550 cm(-1), and their combination tones between 3010 and 3040 cm(-1), are definitively identified with the biring structure involving H284 and Y288 in the E. coli enzyme. Studies of a synthetic analogue of the H-Y structure, 4-methylimidazole covalently linked to p-cresol, show that a feature near 1540 cm(-1) is unique to the biring structure and is absent from the infrared spectrum of 4-methylimidazole or p-cresol alone. This feature is readily detectable by infrared difference techniques, and offers a direct spectroscopic probe for potential radical production involving the H-Y structure in the O(2) reduction cycle of the oxidases.
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Farol
authors.initials: F
authors.lastname: Tomson
authors.forename: James A
authors.initials: JA
authors.lastname: Bailey
authors.forename: Robert B
authors.initials: RB
authors.lastname: Gennis
authors.forename: Clifford J
authors.initials: CJ
authors.lastname: Unkefer
authors.forename: Zizhong
authors.initials: Z
authors.lastname: Li
authors.forename: Louis A
authors.initials: LA
authors.lastname: Silks
authors.forename: Rodolfo A
authors.initials: RA
authors.lastname: Martinez
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Donohoe
authors.forename: R Brian
authors.initials: RB
authors.lastname: Dyer
authors.forename: William H
authors.initials: WH
authors.lastname: Woodruff
mesh_terms: D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000138:chemical synthesis / Q000737:chemistry; D005609:Free Radicals / Q000737:chemistry; D006420:Hemeproteins / Q000138:chemical synthesis / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D010088:Oxidoreductases / Q000138:chemical synthesis / Q000737:chemistry*; D013055:Spectrophotometry, Infrared / Q000379:methods; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D005609:Free Radicals; D006420:Hemeproteins; C083850:cytochrome bo3, E coli; D014443:Tyrosine; D006639:Histidine; D002248:Carbon Monoxide; D010088:Oxidoreductases; C024748:copper oxidase