Stabilization Mechanism of Cytochrome c₅₅₂ from a Moderately Thermophilic Bacterium, Hydrogenophilus thermoluteolus

Abstract

Cytochrome c₅₅₂ (PH c₅₅₂) from moderately thermophilic Hydrogenophilus thermoluteolus exhibits stability intermediate between those of cytochrome c₅₅₂ (HT c₅₅₂) from thermophilic Hydrogenobacter thermophilus and cytochrome c₅₅₁ (PA c₅₅₁) from mesophilic Pseudomonas aeruginosa. To understand the mechanism of stabilization of PH c₅₅₂, we introduced mutations into PH c₅₅₂ at five sites, which, in HT c₅₅₂, are occupied by the amino acids responsible for stability higher than the less stable PA c₅₅₁. When PH c₅₅₂ Val-78 was mutated to Ile, as found in HT c₅₅₂, the resulting variant showed increased stability. Mutation of Ala-7, Met-13, and Tyr-34 to the corresponding residues in PA c₅₅₁ (Phe, Val, and Phe, respectively) resulted in destabilization. We also found that PH c₅₅₂ 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 c₅₅₂ is due to the amino acids at these five sites.