title: Role of heme types in heme-copper oxidases: effects of replacing a heme b with a heme o mimic in an engineered heme-copper center in myoglobin.
abstract: To address the role of the secondary hydroxyl group of heme a/o in heme-copper oxidases, we incorporated Fe(III)-2,4 (4,2) hydroxyethyl vinyl deuterioporphyrin IX, as a heme o mimic, into the engineered heme-copper center in myoglobin (sperm whale myoglobin L29H/F43H, called Cu(B)Mb). The only difference between the heme b of myoglobin and the heme o mimic is the substitution of one of the vinyl side chains of the former with a hydroxyethyl group of the latter. This substitution resulted in an approximately 4 nm blue shift in the Soret band and approximately 20 mV decrease in the heme reduction potential. In a control experiment, the heme b in Cu(B)Mb was also replaced with a mesoheme, which resulted in an approximately 13 nm blue shift and approximately 30 mV decrease in the heme reduction potential. Kinetic studies of the heme o mimic-substituted Cu(B)Mb showed significantly different reactivity toward copper-dependent oxygen reduction from that of the b-type Cu(B)Mb. In reaction with O2, Cu(B)Mb with a native heme b showed heme oxygenase activity by generating verdoheme in the presence of Cu(I). This heme degradation reaction was slowed by approximately 19-fold in the heme o mimic-substituted Cu(B)Mb (from 0.028 s(-1) to 0.0015 s(-1)), while the mesoheme-substituted Cu(B)Mb shared a similar heme degradation rate with that of Cu(B)Mb (0.023 s(-1)). No correlation was found between the heme reduction potential and its O2 reactivity. These results strongly suggest the critical role of the hydroxyl group of heme o in modulating heme-copper oxidase activity through participation in an extra hydrogen-bonding network.
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Ningyan
authors.initials: N
authors.lastname: Wang
authors.forename: Xuan
authors.initials: X
authors.lastname: Zhao
authors.forename: Yi
authors.initials: Y
authors.lastname: Lu
mesh_terms: D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D006418:Heme / Q000737:chemistry* / Q000502:physiology; D007700:Kinetics; D008297:Male; D008958:Models, Molecular; D018716:Molecular Mimicry; D015394:Molecular Structure; D009211:Myoglobin / Q000737:chemistry*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering
chemical_list: D009211:Myoglobin; C069808:heme O; D006418:Heme; D010088:Oxidoreductases; C024748:copper oxidase