Local conformational transition of Hydrogenobacter thermophilus cytochrome c552 relevant to its redox potential.

In order to elucidate the molecular mechanisms responsible for the apparent nonlinear behavior of the temperature dependence of the redox potential of Hydrogenobacter thermophilus cytochrome c552 [Takahashi, Y., Sasaki, H., Takayama, S. J., Mikami, S., Kawano, S., Mita, H., Sambongi, Y., and Yamamoto, Y. (2006) Biochemistry 45, 11005-11011], its heme active site structure has been characterized using variable-temperature and -pressure NMR techniques. The study revealed a temperature-dependent conformational transition between protein structures, which slightly differ in the conformation of the loop bearing the Fe-bound axial Met residue. The heme environment in the protein structure which arises at lower temperature was found to be more polar, as a result of the altered orientation of the loop with respect to the heme due to its conformational change, than that arising at higher temperature. The present study demonstrated the importance of the structural and dynamic properties of the polypeptide chain in close proximity to the heme for redox regulation of the protein.

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.