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J. Biol. Chem., Vol. 283, Issue 7, 3708-3717, February 15, 2008

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Interaction between Substrate and Oxygen Ligand Responsible for Effective O–O Bond Cleavage in Bovine Cytochrome P450 Steroid 21-Hydroxylase Proved by Raman Spectroscopy^*

Takehiko Tosha¹, Norio Kagawa², Miharu Arase, Michael R. Waterman, and Teizo Kitagawa^¶3

From the Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan, the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, and the ^¶Toyota Physical and Chemical Research Institute, Nagakute, Aichi 480-1192, Japan

We investigated structural and functional properties of bovine cytochrome P450 steroid 21-hydroxylase (P450c21), which catalyzes hydroxylation at C-21 of progesterone and 17-hydroxyprogesterone. The uncoupled H₂O₂ formation was higher in the hydroxylation of progesterone (26% of NADPH consumed) than that of 17-hydroxyprogesterone (15% of NADPH consumed), indicating that 17-hydroxyprogesterone can better facilitate the O–O bond scission. In relation to this, it is noted that the O–O stretching mode (_O–O) of the oxygen complex of P450c21 was sensitive to the substrate; the progesterone- or 17-hydroxyprogesterone-bound enzyme gave single (at 1137 cm^–1) or split _O–O bands (at 1124 and 1138 cm^–1), respectively, demonstrating the presence of two forms for the latter. In contrast to _O–O, no corresponding difference was observed for the Fe-O₂ stretching mode between two different substrate-bound forms. The Fe-S(Cys) stretching mode in the ferric state was also identical (349 cm^–1) for each substrate-bound form, suggesting that modulation through the axial thiolate by the substrate is unlikely. Therefore, it is deduced that the hydroxyl group at C-17 of 17-hydroxyprogesterone forms a hydrogen bond with the terminal oxygen atom of the FeOO complex in one form, yielding a lower _O–O frequency with higher reactivity for O–O cleavage, whereas the other form in which the substrate does not provide a hydrogen bond to the oxygen ligand is essentially the same between the two kinds of substrates. In the hydrogen-bonded species, the substrate changes the geometry of the FeOO moiety, thereby performing the hydroxylation reaction more effectively in 17-hydroxyprogesterone than in progesterone.

Received for publication, August 31, 2007

^* This work was supported by Grant-in-aid for Specifically Promoted Research 14001004 (to T. K.) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and National Institutes of Health Grants GM37942 and ES00267. 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 Supported by a research fellowship for young scientists from the Japan Society for the Promotion of Science. Present address: Children's Hospital Oakland Research Institute, Oakland, CA 94609.

² Present address: Dept. of Biochemistry, Saarland University, 66123 Saarbrucken, Germany.

³ To whom correspondence should be addressed. Tel.: 81-80-1620-8159; Fax: 81-564-63-6302; E-mail: Riken-kitagawa{at}mosk.tytlabs.co.jp.

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