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

Abstract

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.

• cytochrome c
• electrostatic interaction
• protein stability
• site-directed mutagenesis