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 O2 at 1558 cm -1 and O3 at 1471 cm -1 . O4 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 OFe-CN line at 446 cm -1 and ‰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: OFe-CO (531 cm -1 ), ‰Fe-C-O (567 cm -1 ), and OC-O (1954 cm -1 ). No hydrogen bonding was detected for the heme-bound CO in PGHS-1. These frequencies markedly deviated from the OFe-CO/OC-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 (OFe-CO: 496 cm -1 ) where disruption of the hydrogen bonding interactions to the proximal histidine may occur.