Peroxidase activity in prostaglandin endoperoxide H synthase-1 occurs with a neutral histidine proximal heme ligand

Steve A. Seibold, Jose F. Cerda, Anne M. Mulichak, Inseok Song, R. Michael Garavito, Toshiya Arakawa, William L. Smith, Gerald T. Babcock

Research output: Contribution to journalArticlepeer-review

30 Scopus citations


Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) convert arachidonic acid to prostaglandin H2 (PGH2), the committed step in prostaglandin and thromboxane formation. Interaction of peroxides with the heme sites in PGHSs generates a tyrosyl radical that catalyzes subsequent cyclooxygenase chemistry. To study the peroxidase reaction of ovine oPGHS-1, we combined spectroscopic and directed mutagenesis data with X-ray crystallographic refinement of the heme site. Optical and Raman spectroscopy of oxidized oPGHS-1 indicate that its heme iron (Fe3+) exists exclusively as a high-spin, six-coordinate species in the holoenzyme and in heme- reconstituted apoenzyme. The sixth ligand is most likely water. The cyanide complex of oxidized oPGHS-1 has a six-coordinate, low-spin ferric iron with a v[Fe-CN] frequency at 445 cm-1; a monotonic sensitivity to cyanide isotopomers that indicates the FeCN adduct has a linear geometry. The ferrous iron in reduced oPGHS-1 adopts a high, spin, five-coordinate state that is converted to a six-coordinate, low-spin geometry by CO. The low-frequency Raman spectrum of reduced oPGHS-1 reveals two v[Fe-His] frequencies at 206 and 222 cm-1. These vibrations, which disappear upon addition of CO, are consistent with a neutral histidine (His388) as the proximal heme ligand. The refined crystal structure shows that there is a water molecule located between His388 and Tyr504 that can hydrogen bond to both residues. However, substitution of Tyr504 with alanine yields a mutant having 46% of the peroxidase activity of native oPGHS-1, establishing that bonding of Tyr504 to this water is not critical for catalysis. Collectively, our results show that the proximal histidine ligand in oPGHS-1 is electrostatically neutral. Thus, in contrast to most other peroxidases, a strongly basic proximal ligand is not necessary for peroxidase catalysis by oPGHS-1.

Original languageEnglish
Pages (from-to)6616-6624
Number of pages9
Issue number22
StatePublished - 6 Jun 2000


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