Hyperstability and crystal structure of cytochrome c₅₅₅ from hyperthermophilic Aquifex aeolicus

In order to elucidate the relationship between the stability and the structure of the monohaem cytochrome c₅₅₅ (AA c₅₅₅) from the hyperthermophilic bacterium Aquifex aeolicus, chemical denaturation and crystal structure determination were carried out. AA c₅₅₅ exhibited higher stability than the thermophilic Hydrogenobacter thermophilus cytochrome c₅₅₂ (HT c₅₅₂), which is one of the most stable cytochromes c. The three-dimensional crystal structure of AA c₅₅₅, which was determined using the multiple anomalous dispersion technique at 1.15 Å resolution, included a unique 14-residue extra helix, while the side-chain inter­actions of several amino-acid residues responsible for the stability of HT c₅₅₂ were conserved in AA c₅₅₅. The side chain of the Met61 residue in the extra helix was aligned towards the haem, forming a coordination bond between the Met S and haem Fe atoms. In other cytochromes c the corresponding regions always form Ω loops which also include the haem-liganding Met residue and are known to be involved in the initial step in cytochrome c denaturation. The formation of the extra helix in AA c₅₅₅ results in the highest helix content, 59.8%, among the monohaem cytochromes c. The extra helix should mainly contribute to the hyperstability of AA c₅₅₅ and is presumed to be a novel strategy of cytochromes c for adaptation to a hyperthermophilic environment.