Further enhancement of the thermostability of Hydrogenobacter thermophilus cytochrome c552.
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.
Takahashi
Yo-ta
YT
Department of Chemistry, University of Tsukuba, Tsukuba 305-8571, Japan.
Sasaki
Hiroaki
H
Takayama
Shin-ichi J
SJ
Mikami
Shin-ichi
S
Kawano
Shin
S
Mita
Hajime
H
Sambongi
Yoshihiro
Y
Yamamoto
Yasuhiko
Y
D019943:Amino Acid Substitution; D001419:Bacteria / Q000201:enzymology*; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013696:Temperature
D003574:Cytochrome c Group; C022302:cytochrome C-552