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

Cytochrome csub(552) (PH csub(552)) from moderately thermophilic Hydrogenophilus thermoluteolus exhibits stability intermediate between those of cytochrome csub(552) (HT csub(552)) from thermophilic Hydrogenobacter thermophilus and cytochrome c551 (PA csub(551)) from mesophilic Pseudomonas aeruginosa. To understand the mechanism of stabilization of PH csub(552), we introduced mutations into PH csub(552) at five sites, which, in HT csub(552), are occupied by the amino acids responsible for stability higher than the less stable PA csub(551). When PH csub(552) Val-78 was mutated to Ile, as found in HT csub(552), the resulting variant showed increased stability. Mutation of Ala-7, Met-13, and Tyr-34 to the corresponding residues in PA csub(551) (Phe, Val, and Phe, respectively) resulted in destabilization. We also found that PH csub(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 csub(552) is due to the amino acids at these, five sites.