Further enhancement of the thermostability of Hydrogenobacter thermophilus cytochrome c552

Thermophile Hydrogenobacter thermophilus cytochrome c552 ( 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 similar to 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 similar to 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.