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J. Biol. Chem., Vol. 282, Issue 22, 16681-16690, June 1, 2007

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Unique Peroxidase Reaction Mechanism in Prostaglandin Endoperoxide H Synthase-2

COMPOUND I IN PROSTAGLANDIN ENDOPEROXIDE H SYNTHASE-2 CAN BE FORMED WITHOUT ASSISTANCE BY DISTAL GLUTAMINE RESIDUE^*

Shizuo Ichimura, Takeshi Uchida^¶||, Shuhei Taniguchi, Shusuke Hira, Takehiko Tosha^¶, Isao Morishima, Teizo Kitagawa^¶1, and Koichiro Ishimori^||**2

From the Division of Chemistry, Graduate School of Science, Hokkaido University, Hokkaido 060-0810, Japan, Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan, ^¶Okazaki Institute for Integrative Bioscience, Okazaki 444-8787, Japan, ^||Division of Chemistry, Faculty of Science, Hokkaido University, Hokkaido 060-0810, Japan, and ^**Institute for Molecular Science, Okazaki 444-8585, Japan

Prostaglandin-endoperoxide H synthase-2 (PGHS-2) shows peroxidase activity to promote the cyclooxygenase reaction for prostaglandin H₂, but one of the highly conserved amino acid residues in peroxidases, distal Arg, stabilizing the developing negative charge on the peroxide through a hydrogen-bonding interaction, is replaced with a neutral amino acid residue, Gln. To characterize the peroxidase reaction in PGHS-2, we prepared three distal glutamine (Gln-189) mutants, Arg (GlnArg), Asn (Gln Asn), and Val (Gln Val) mutants, and examined their peroxidase activity together with their structural characterization by absorption and resonance Raman spectra. Although a previous study (Landino, L. M., Crews, B. C., Gierse, J. K., Hauser, S. D., and Marnett, L. (1997) J. Biol. Chem. 272, 21565-21574) suggested that the Gln residue might serve as a functionally equivalent residue to Arg, our current results clearly showed that the peroxidase activity of the Val and Asn mutants was comparable with that of the wild-type enzyme. In addition, the Fe-C and C-O stretching modes in the CO adduct were almost unperturbed by the mutation, implying that Gln-189 might not directly interact with the heme-ligated peroxide. Rather, the peroxidase activity of the Arg mutant was depressed, concomitant with the heme environmental change from a six-coordinate to a five-coordinate structure. Introduction of the bulky amino acid residue, Arg, would interfere with the ligation of a water molecule to the heme iron, suggesting that the side chain volume, and not the amide group, at position 189 is essential for the peroxidase activity of PGHS-2. Thus, we can conclude that the O-O bond cleavage in PGHS-2 is promoted without interactions with charged side chains at the peroxide binding site, which is significantly different from that in typical plant peroxidases.

Received for publication, November 21, 2006 , and in revised form, March 30, 2007.

^* This work was supported in part by Grants-in-aid 15350101 (to K. I.) from the Ministry of Education, Culture, Science, Sports, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Toyota Physical & Chemical Research Inst., Nagakute, Aichi 480-1192, Japan.

² To whom correspondence should be addressed: Division of Chemistry, Faculty of Science, Hokkaido University, Hokkaido 060-0810, Japan. Tel.: 81-11-706-2707; Fax: 81-11-706-3501; E-mail: koichiro{at}sci.hokudai.ac.jp.

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