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J. Biol. Chem., Vol. 280, Issue 10, 9129-9134, March 11, 2005

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Rational Design of a Mononuclear Metal Site into the Archaeal Rieske-type Protein Scaffold^*

Toshio Iwasaki, Asako Kounosu, Ye Tao¶||, Zhongrui Li¶**, Jacob E. Shokes¶, Nathaniel J. Cosper¶, Takeo Imai, Akio Urushiyama, and Robert A. Scott¶

From the Department of Biochemistry and Molecular Biology, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan, the ^¶Department of Chemistry, University of Georgia, Athens, Georgia 30602-2556, the ^||Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100039, China, and the Department of Chemistry, Rikkyo (St. Paul's) University, Toshima-ku, Tokyo 171-8501, Japan

Proteins containing Rieske-type [2Fe-2S] clusters play essential functions in all three domains of life. We engineered the two histidine ligands to the Rieske-type [2Fe-2S] cluster in the hyperthermophilic archaeal Rieske-type ferredoxin from Sulfolobus solfataricus to modify types and spacing of ligands and successfully converted the metal and cluster type at the redox-active site with a minimal structural change to a native Rieske-type protein scaffold. Spectroscopic analyses unambiguously established a rubredoxin-type mononuclear Fe^3+/2+ center at the engineered local metal-binding site (Zn²⁺ occupies the iron site depending on the expression conditions). These results show the importance of types and spacing of ligands in the in vivo cluster recognition/insertion/assembly in biological metallosulfur protein scaffolds. We suggest that early ligand substitution and displacement events at the local metal-binding site(s) might have primarily allowed the metal and cluster type conversion in ancestral redox protein modules, which greatly enhanced their capabilities of conducting a wide range of unique redox chemistry in biological electron transfer conduits, using a limited number of basic protein scaffolds.

Received for publication, December 14, 2004 , and in revised form, December 28, 2004.

^* This work was supported by the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) Grant-in-aid 15770088 (to T. Iwasaki), by Japanese Society for the Promotion of Science (JSPS) Grant BSAR-507 (to T. Iwasaki), by Grant GM 42025 from the National Institutes of Health (to R. A. S.), and by financial support to the Research Center for Measurement in Advanced Science of Rikkyo (St. Paul's) University. 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.

^** Current address: Dept. of Chemical Engineering and Material Science, University of Oklahoma, Norman, OK 73019.

To whom correspondence may be addressed: Dept. of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyoku, Tokyo 113-8602, Japan. Tel.: 81-3-3822-2131 (ext. 5216); Fax: 81-3-5685-3054; E-mail: tiwasaki{at}nms.ac.jp. To whom correspondence may be addressed: Dept. of Chemistry, University of Georgia, Athens, GA 30602-2556. Tel.: 706-542-2240; Fax: 706-542-2295; E-mail: scott{at}chem.uga.edu.

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