Change in structure and ligand binding properties of hyperstable cytochrome c₅₅₅ from Aquifex aeolicus by domain swapping

Abstract

Cytochrome c 555 from hyperthermophilic bacteria Aquifex aeolicus (AA cyt c 555 ) is a hyperstable protein belonging to the cyt c protein family, which possesses a unique long 3 10 -a-3 10 helix containing the heme-ligating Met61. Herein, we show that AA cyt c 555 forms dimers by swapping the region containing the extra 3 10 -a-3 10 helix and C-terminal a-helix. The asymmetric unit of the crystal of dimeric AA cyt c 555 contained two dimer structures, where the structure of the hinge region (Val53-Lys57) was different among all four protomers. Dimeric AA cyt c 555 dissociated to monomers at 92 6 1 C according to DSC measurements, showing that the dimer was thermostable. According to CD measurements, the secondary structures of dimeric AA cyt c 555 were maintained at pH 2.2-11.0. CN -and CO bound to dimeric AA cyt c 555 in the ferric and ferrous states, respectively, owing to the flexibility of the hinge region close to Met61 in the dimer, whereas these ligands did not bind to the monomer under the same conditions. In addition, CN -and CO bound to the oxidized and reduced dimer at neutral pH and a wide range of pH (pH 2.2-11.0), respectively, in a wide range of temperature (25-85 C), owing to the thermostability and pH tolerance of the dimer. These results show that the ligand binding character of hyperstable AA cyt c 555 changes upon dimerization by domain swapping.

Formation and carbon monoxide‐dependent dissociation of Allochromatium vinosum cytochrome c′ oligomers using domain‐swapped dimers

“…AVCP is the first example of the domain swapping oligomerization of a class II cyt c . Oligomers formed by domain swapping are frequently metastable and dissociate to monomers irreversibly . In these proteins, the stability of domain‐swapped oligomers correlates well to that of its monomer, since most of the interactions in the monomer are reproduced in the domain‐swapped oligomer, although some of the interactions may convert from within a molecule to between protomers.…”

“…[20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] AVCP is the first example of the domain swapping oligomerization of a class II cyt c. Oligomers formed by domain swapping are frequently metastable and dissociate to monomers irreversibly. [31][32][33][34][35][36]53,54 In these proteins, the stability of domain-swapped oligomers correlates well to that of its monomer, since most of the interactions in the monomer are reproduced in the domain-swapped oligomer, although some of the interactions may convert from within a molecule to between protomers. The denaturation temperature of AVCP in its homodimeric form has been reported as 528C, 55 and thus the domain-swapped dimer of the AVCP tetramer may dissociate to monomers relatively easily, producing native dimers (Supporting Information Fig S1).…”

“…These results indicate that oligomeric AVCP was formed by the ethanol treatment, similar to the properties in other c-type cyts. [31][32][33][34]38 The purified tetrameric AVCP converted to native dimers at low salt concentrations, whereas the dissociation was inhibited in the presence of 160 mM sodium sulfate (Supporting Information Fig. S1).…”

“…Class I monomeric globular c-type cytochromes [e.g., horse cytochrome (cyt) c, mesophile Pseudomonas aeruginosa (PA) cyt c 551 , thermophile Hydrogenobacter thermophilus (HT) cyt c 552 , and hyperthermophile Aquifex aeolicus (AA) cyt c 555 ] form domain-swapped oligomers by exchanging the N-or C-terminal region between molecules. [31][32][33][34] The c-type mutant of Escherichia coli cyt b 562 (cyt cb 562 ) exhibiting a four helix bundle structure, as well as horse myoglobin (Mb) possessing a noncovalently bound heme, form domain-swapped dimers by exchanging half of the structure between molecules. 35,36 Domain swapping has also been used to construct various protein oligomers.…”

Domain-swapped cytochrome cb562dimer and its nanocage encapsulating a Zn–SO4cluster in the internal cavity

“…10 We have previously shown that small spherical heme proteins, c -type cytochrome (cyt) proteins and horse myoglobin (Mb), form oligomers by domain swapping. 11 In the dimer and trimer of horse cyt c , the C-terminal α-helix domain swapped with the corresponding region of other molecules. In dimeric Pseudomonas aeruginosa (PA) cyt c 551 and dimeric Hydrogenobacter thermophilus (HT) cyt c 552 , the region containing the N-terminal α-helix and heme was swapped.…”