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

Oikawa Kenta

Nakamura Shota

Sonoyama Takafumi

Ohshima Atsushi

Kobayashi Yuji

Takayama Shin-ichi J.

Yamamoto Yasuhiko

Uchiyama Susumu

Hasegawa Jun

Sambongi Yoshihiro

Journal of Biological Chemistry

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.

eng

The American Society for Biochemistry and Molecular Biology, Inc.

This research was originally published in the Journal of Biological Chemistry. Kenta Oikawa, Shota Nakamura, Takafumi Sonoyama, Atsushi Ohshima, Yuji Kobayashi, Shin-ichi J. Takayama, Yasuhiko Yamamoto, Susumu Uchiyama, Jun Hasegawa, and Yoshihiro Sambongi. Five Amino Acid Residues Responsible for the High Stability of Hydrogenobacter thermophilus Cytochrome c552. J Biol Chem. 2005; 280:5527-5532. © 2005 the American Society for Biochemistry and Molecular Biology, Inc.

[ISSN] 0021-9258

[ISSN] 1083-351X

[DOI] 10.1074/jbc.M412392200

[DOI] https://doi.org/10.1074/jbc.M412392200