Peroxidase Activity in Prostaglandin Endoperoxide H Synthase-1 Occurs with a Neutral Histidine Proximal Heme Ligand

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• 作者：Steve A. Seibold ; Jose F. Cerda ; Anne M. Mulichak ; Inseok Song ; R. Michael Garavito ; Toshiya Arakawa ; William L. Smith ; and Gerald T. Babcock
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：June 6, 2000
• 年：2000
• 卷：39
• 期：22
• 页码：6616 - 6624
• 全文大小：259K
• 年卷期：v.39,no.22(June 6, 2000)
• ISSN：1520-4995

文摘

Prostaglandin endoperoxide H synthases-1 and -2 (PGHS-1 and -2) convert arachidonic acidto prostaglandin H₂ (PGH₂), the committed step in prostaglandin and thromboxane formation. Interactionof peroxides with the heme sites in PGHSs generates a tyrosyl radical that catalyzes subsequentcyclooxygenase chemistry. To study the peroxidase reaction of ovine oPGHS-1, we combined spectroscopicand directed mutagenesis data with X-ray crystallographic refinement of the heme site. Optical and Ramanspectroscopy 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 mostlikely water. The cyanide complex of oxidized oPGHS-1 has a six-coordinate, low-spin ferric iron witha v[Fe-CN] frequency at 445 cm^-1; a monotonic sensitivity to cyanide isotopomers that indicates theFe-CN 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 Ramanspectrum 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 proximalheme ligand. The refined crystal structure shows that there is a water molecule located between His388and Tyr504 that can hydrogen bond to both residues. However, substitution of Tyr504 with alanine yieldsa mutant having 46% of the peroxidase activity of native oPGHS-1, establishing that bonding of Tyr504to this water is not critical for catalysis. Collectively, our results show that the proximal histidine ligandin oPGHS-1 is electrostatically neutral. Thus, in contrast to most other peroxidases, a strongly basic proximalligand is not necessary for peroxidase catalysis by oPGHS-1.