title: Resonance Raman studies indicate a unique heme active site in prostaglandin H synthase.
abstract: Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) catalyze the first two steps in the biosynthesis of prostaglandins. Resonance Raman spectroscopy was used to characterize the PGHS heme active site and its immediate environment. Ferric PGHS-1 has a predominant six-coordinate high-spin heme at room temperature, with water as the sixth ligand. The proximal histidine ligand (or the distal water ligand) of this hexacoordinate high-spin heme species was reversibly photolabile, leading to a pentacoordinate high-spin ferric heme iron. Ferrous PGHS-1 has a single species of five-coordinate high-spin heme, as evident from nu(2) at 1558 cm(-1) and nu(3) at 1471 cm(-1). nu(4) at 1359 cm(-1) indicates that histidine is the proximal ligand. A weak band at 226-228 cm(-1) was tentatively assigned as the Fe-His stretching vibration. Cyanoferric PGHS-1 exhibited a nu(Fe)(-)(CN) line at 446 cm(-1) and delta(Fe)(-)(C)(-)(N) at 410 cm(-1), indicating a "linear" Fe-C-N binding conformation with the proximal histidine. This linkage agrees well with the open distal heme pocket in PGHS-1. The ferrous PGHS-1 CO complex exhibited three important marker lines: nu(Fe)(-)(CO) (531 cm(-1)), delta(Fe)(-)(C)(-)(O) (567 cm(-1)), and nu(C)(-)(O) (1954 cm(-1)). No hydrogen bonding was detected for the heme-bound CO in PGHS-1. These frequencies markedly deviated from the nu(Fe)(-)(CO)/nu(C)(-)(O) correlation curve for heme proteins and porphyrins with a proximal histidine or imidazolate, suggesting an extremely weak bond between the heme iron and the proximal histidine in PGHS-1. At alkaline pH, PGHS-1 is converted to a second CO binding conformation (nu(Fe)(-)(CO): 496 cm(-1)) where disruption of the hydrogen bonding interactions to the proximal histidine may occur.
authors.affiliation: Novartis Pharmaceuticals Corporation, 556 Morris Avenue, Summit, New Jersey 07901, USA.
authors.forename: B S
authors.initials: BS
authors.lastname: Lou
authors.forename: J K
authors.initials: JK
authors.lastname: Snyder
authors.forename: P
authors.initials: P
authors.lastname: Marshall
authors.forename: J S
authors.initials: JS
authors.lastname: Wang
authors.forename: G
authors.initials: G
authors.lastname: Wu
authors.forename: R J
authors.initials: RJ
authors.lastname: Kulmacz
authors.forename: A L
authors.initials: AL
authors.lastname: Tsai
authors.forename: J
authors.initials: J
authors.lastname: Wang
mesh_terms: D000818:Animals; D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry; D003486:Cyanides / Q000737:chemistry; D005290:Ferric Compounds / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D008024:Ligands; D008297:Male; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D012756:Sheep; D013059:Spectrum Analysis, Raman
chemical_list: D003486:Cyanides; D005290:Ferric Compounds; D005296:Ferrous Compounds; D008024:Ligands; D006418:Heme; D002248:Carbon Monoxide; D007501:Iron; D011451:Prostaglandin-Endoperoxide Synthases