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J. Biol. Chem., Vol. 280, Issue 7, 5527-5532, February 18, 2005

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Five Amino Acid Residues Responsible for the High Stability of Hydrogenobacter thermophilus Cytochrome c₅₅₂

RECIPROCAL MUTATION ANALYSIS^*

Kenta Oikawa, Shota Nakamura, Takafumi Sonoyama, Atsushi Ohshima, Yuji Kobayashi, Shin-ichi J. Takayama¶, Yasuhiko Yamamoto¶, Susumu Uchiyama||, Jun Hasegawa**, and Yoshihiro Sambongi

From the Graduate School of Biosphere Science, Hiroshima University, CREST of Japan Science and Technology Corp., 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan, Graduate School of Pharmaceutical Sciences, Osaka University, Suita 565-0871, Japan, ^¶Department of Chemistry, University of Tsukuba, Tsukuba 305-8571, Japan, ^||Graduate School of Engineering, Osaka Univeristy, Suita 565-0871, Japan, and ^**Daiichi Pharmaceutical Co., Ltd., 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan

Five amino acid residues responsible for extreme stability have been identified in cytochrome c₅₅₂ (HT c₅₅₂) from a thermophilic bacterium, Hydrogenobacter thermophilus. The five residues, which are spatially distributed in three regions of HT c₅₅₂, were replaced with the corresponding residues in the homologous but less stable cytochrome c₅₅₁ (PA c₅₅₁) from Pseudomonas aeruginosa. The quintuple HT c₅₅₂ variant (A7F/M13V/Y34F/Y43E/I78V) showed the same stability against guanidine hydrochloride denaturation as that of PA c₅₅₁, suggesting that the five residues in HT c₅₅₂ necessarily and sufficiently contribute to the overall stability. In the three HT c₅₅₂ variants carrying mutations in each of the three regions, the Y34F/Y43E mutations resulted in the greatest destabilization, by –13.3 kJ mol^–1, followed by A7F/M13V (–3.3 kJ mol^–1) and then I78V (–1.5 kJ mol^–1). The order of destabilization in HT c₅₅₂ was the same as that of stabilization in PA c₅₅₁ with reverse mutations such as F34Y/E43Y, F7A/V13M, and V78I (13.4, 10.3, and 0.3 kJ mol^–1, respectively). The results of guanidine hydrochloride denaturation were consistent with those of thermal denaturation for the same variants. The present study established a method for reciprocal mutation analysis. The effects of side-chain contacts were experimentally evaluated by swapping the residues between the two homologous proteins that differ in stability. A comparative study of the two proteins was a useful tool for assessing the amino acid contribution to the overall stability.

Received for publication, November 2, 2004 , and in revised form, December 6, 2004.

^* This work was supported in part by grants from Hiroshima University, the Noda Institute for Scientific Research, and the Japanese Ministry of Education, Science and Culture (grants-in-aid for Scientific Research on Priority Areas). 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.

To whom correspondence should be addressed: Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan. Tel. and Fax: 81-82-424-7924; E-mail: sambongi{at}hiroshima-u.ac.jp.

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