Hyperstability and crystal structure of cytochromec₅₅₅from hyperthermophilicAquifex aeolicus

Abstract

In order to elucidate the relationship between the stability and the structure of the monohaem cytochrome c(555) (AA c(555)) from the hyperthermophilic bacterium Aquifex aeolicus, chemical denaturation and crystal structure determination were carried out. AA c(555) exhibited higher stability than the thermophilic Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), which is one of the most stable cytochromes c. The three-dimensional crystal structure of AA c(555), which was determined using the multiple anomalous dispersion technique at 1.15 A resolution, included a unique 14-residue extra helix, while the side-chain interactions of several amino-acid residues responsible for the stability of HT c(552) were conserved in AA c(555). 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 Omega 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 c(555) results in the highest helix content, 59.8%, among the monohaem cytochromes c. The extra helix should mainly contribute to the hyperstability of AA c(555) and is presumed to be a novel strategy of cytochromes c for adaptation to a hyperthermophilic environment.

High stability of apo-cytochrome c’ from thermophilic Hydrogenophilus thermoluteolus

“…8) Therefore, PHCP may be produced through DsbD (also known as DipZ, functioning as a reductant for cytochrome c heme-binding Cys residues 9) )-independent biosynthesis in E. coli. 2) Consistently, similar DsbD-independent biosynthesis has been observed exceptionally for hyperthermophilic Aquifex aeolicus class I cytochrome c 555 (AA cytc 555 ), 10,11) which is also structured as an apo-protein. 12) Thermal stability of the apo-PHCP protein (20 μM) in 20 mM potassium phosphate (pH 7.0) was then measured by the temperature-dependent CD ellipticity change at 222 nm.…”

Heme Is Not Required forAquifex aeolicusCytochromec555Polypeptide Folding

“…Recently, we determined the three-dimensional structure of holo AA c 555 , 23) in which most of these hydrophobic residues are localized in the helical structures (Fig. 4) and constructing a hydrophobic core in the protein interior.…”

Effects of heme on the thermal stability of mesophilic and thermophilic cytochromes c: Comparison between experimental and theoretical results

“…In order to examine the packing efficiency of the apo polypeptide, we calculate the measure S VH,25,EX /(k B N r ) for the imaginary transition illustrated in Fig. This result must closely be related to the following experimental observation: [3][4][5][6] In the apo states of PA c 551 , PH c 552 , and HT c 552 , almost all helices are collapsed at 25 • C whereas the α-helix content of the apo state of AA c 555 is almost equal to that of the holo state. That is, heme is not incorporated in the calculation.…”

“…Sambongi and co-workers [3][4][5] have experimentally performed comparative studies for cytochrome c551 from Pseudomonas aeruginosa (PA c 551 ), cytochrome c552 from Pseudomonas hydrogenothermophila (PH c 552 ), cytochrome c552 from Hydrogenobacter thermophilus (HT c 552 ), and cytochrome c555 from Aquifex aeolicus (AA c 555 ). PA c 551 , PH c 552 , HT c 552 , and AA c 555 are models of mesophilic, moderately thermophilic, thermophilic, and hyperthermophilic proteins, respectively.…”