Stabilization mechanism of cytochrome c552 from a moderately thermophilic bacterium, Hydrogenophilus thermoluteolus.

Cytochrome c(552) (PH c(552)) from moderately thermophilic Hydrogenophilus thermoluteolus exhibits stability intermediate between those of cytochrome c(552) (HT c(552)) from thermophilic Hydrogenobacter thermophilus and cytochrome c(551) (PA c(551)) from mesophilic Pseudomonas aeruginosa. To understand the mechanism of stabilization of PH c(552), we introduced mutations into PH c(552) at five sites, which, in HT c(552), are occupied by the amino acids responsible for stability higher than the less stable PA c(551). When PH c(552) Val-78 was mutated to Ile, as found in HT c(552), the resulting variant showed increased stability. Mutation of Ala-7, Met-13, and Tyr-34 to the corresponding residues in PA c(551) (Phe, Val, and Phe, respectively) resulted in destabilization. We also found that PH c(552) Lys-43 contributed to stability through the formation of an attractive electrostatic interaction with Asp-39. These results suggest that the intermediate stability of PH c(552) is due to the amino acids at these five sites.