Resonance raman studies indicate a unique heme active site in prostaglandin H synthase

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 v₂ at 1558 cm_-1 and v₃ at 1471 cm^-1. v₄ 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 v(Fe-CN) line at 446 cm^-1 and δ(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: v(Fe-CO) (531 cm^-1), δ(Fe-C-O) (567 cm^-1), and v(C-O) (1954 cm^-1). No hydrogen bonding was detected for the heme-bound CO in PGHS-1. These frequencies markedly deviated from the v(Fe-CO/v(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 (v(Fe-CO): 496 cm^-1) where disruption of the hydrogen bonding interactions to the proximal histidine may occur.