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Communication

Evaluation of the Functional Role of the Heme-6-propionate Side Chain in Cytochrome P450cam

Supporting Info

Katsuyoshi Harada,^† Keisuke Sakurai,^‡ Kenichiro Ikemura,^§ Takashi Ogura,^§ Shun Hirota, Hideo Shimada,*^# and Takashi Hayashi*^†

Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan, Institute for Protein Research, Osaka University, Suita 565-0871, Japan, Graduate School of Life Science, University of Hyogo, Ako 678-1297, Japan, Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma 630-0192, Japan, and Department of Biochemistry, School of Medicine, Keio University, Tokyo 160-8582, Japan

J. Am. Chem. Soc., 2008, 130 (2), pp 432–433

DOI: 10.1021/ja077902l

Publication Date (Web): December 19, 2007

†

Department of Applied Chemistry, Osaka University.

‡

Institute for Protein Research, Osaka University.

University of Hyogo.

Nara Institute for Science and Technology.

In papers with more than one author, the asterisk indicates the name of the author to whom inquiries about the paper should be addressed.

Keio University.

, thayashi@chem.eng.osaka-u.ac.jp

Abstract

Cytochrome P450cam (P450cam) binds a protoheme IX as a prosthetic group via noncovalent interactions. Heme-6-propionate, one of the two heme-propionate side chains, forms hydrogen-bonding interactions with Arg112 and other hydrophilic amino acid residues. Here, we demonstrate the structural and functional roles of the 6-propionate side chain in P450cam using a reconstituted protein with 6-depropionate-6-methylated protoheme IX (one-legged heme). The spectroscopic data and the enzymatic activities reveal that removal of the 6-propionate has no clear influence on the enzyme property. The rate of electron transfer from putidaredoxin (Pdx), a natural redox partner, to P450cam was not significantly changed, whereas, the removal of the 6-propionate decreased the affinity of Pdx by 3.5-fold supporting the proposed role of Arg112 as the essential constituent of the Pdx binding site. Resonance Raman experiments indicate that removal of the 6-propionate weakens the Fe−S bond strength. The X-ray structure of the reconstituted protein at 1.55 Å resolution is highly superimposable with that of the wild-type protein, whereas the thiolate of the Cys357 heme ligand in the reconstituted protein is visible from the protein surface owing to the lack of the 6-propionate. Lengthening of the Fe−S bond and the water accessibility could facilitate protonation of thiolate anion to thiol, explaining the observed formation of the inactive P420 species under the mild conditions. Therefore, the d-camphor hydroxylation reaction requires a 6-propionate−protein matrix interaction to maintain an active P450 species.

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