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 firs t two steps in the biosynthesis of prostaglandins. Resonance Raman spectros copy was used to characterize the PGHS heme active site and its immediate e nvironment. Ferric PGHS-1 has a predominant six-coordinate high-spin heme a t room temperature, with water as the sixth ligand. The proximal histidine ligand (or the distal water ligand) of this hexacoordinate high-spin heme s pecies was reversibly photolabile, leading to a pentacoordinate high-spin f erric heme iron. Ferrous PGHS-1 has a single species of five-coordinate hig h-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 we ak 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 conformat ion with the proximal histidine, This linkage agrees well with the open dis tal heme pocket in PGHS-1, The ferrous PGHS-1 CO complex exhibited three im portant marker lines: nu(Fe-co) (531 cm(-1)), delta(Fe-C-O) (567 cm(-1)), a nd nu(C-O) (1954 cm(-1)). No hydrogen bonding was detected for the heme-bou nd 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 his tidine or imidazolate, suggesting an extremely weak bond between the heme i ron and the proximal histidine in PGHS-1. At alkaline pH, PGHS-1 is convert ed to a second CO binding conformation (nu(Fe-CO): 496 cm(-1)) where disrup tion of the hydrogen bonding interactions to the proximal histidine may occ ur.