Further Enhancement of the Thermostability of Hydrogenobacter thermophilus Cytochrome c552

• Published in: Biochemistry (Easton) Vol. 45; no. 36; pp. 11005 - 11011
• Main Authors: TAKAHASHI, Yo-ta, SASAKI, Hiroaki, TAKAYAMA, Shin-ichi J., MIKAMI, Shin-ichi, KAWANO, Shin, MITA, Hajime, SAMBONGI, Yoshihiro, YAMAMOTO, Yasuhiko
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 12.09.2006
• Subjects: Amino Acid Substitution; Bacteria - enzymology; Circular Dichroism; Cytochrome c Group - chemistry; Cytochrome c Group - genetics; Enzyme Stability; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Magnetic Resonance Spectroscopy; Models, Molecular; Mutation; Oxidation-Reduction; Protein Conformation; Temperature
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi061164g

Thermophile Hydrogenobacter thermophilus cytochrome c(552) (HT) is a stable protein with denaturation temperatures (T(m)) of 109.8 and 129.7 degrees C for the oxidized and reduced forms, respectively [Uchiyama, S., Ohshima, A., Nakamura, S., Hasegawa, J., Terui, N., Takayama, S. J., Yamamoto, Y., Sambongi, Y., and Kobayashi, Y. (2004) J. Am. Chem. Soc. 126, 14684-14685]. The removal of a single hydroxyl group from the hydrophobic core of HT, through the replacement of a Tyr by Phe, resulted in further elevation of the T(m) value of the oxidized form by approximately 6 degrees C, the T(m) value of the reduced one remaining essentially unaltered. As a result, the redox potential of the mutant with higher stability in the oxidized form exhibited a negative shift of approximately 20 mV relative to that of wild-type HT in an enthalpic manner. These findings indicated that the redox function of a protein can be enthalpically regulated through the stability of the oxidized form by altering the contextual stereochemical packing of hydrophobic residues in the protein interior using protein engineering.