Roles of a short connecting disulfide bond in the stability and function of psychrophilic Shewanella violacea cytochrome c (5).
Cys-59 and Cys-62, forming a disulfide bond in the four-residue loop of Shewanella violacea cytochrome c (5) (SV cytc (5)), contribute to protein stability but not to redox function. These Cys residues were substituted with Ala in SV cytc (5), and the structural and functional properties of the resulting C59A/C62A variant were determined and compared with those of the wild-type. The variant had similar features to those of the wild-type in absorption, circular dichroic, and paramagnetic (1)H NMR spectra. In addition, the redox potentials of the wild-type and variant were essentially the same, indicating that removal of the disulfide bond from SV cytc (5) does not affect the redox function generated in the vicinity of heme. However, calorimetric analysis of the wild-type and variant showed that the mutations caused a drastic decrease in the protein stability through enthalpy, but not entropy. Four residues are encompassed by the SV cytc (5) disulfide bond, which is the shortest one that has been proved to affect protein stability. The protein stability of SV cytc (5) can be controlled without changing the redox function, providing a new strategy for regulating the stability and function of cytochrome c.
Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, 739-8528, Japan.
Keiko
K
Ogawa
Takafumi
T
Sonoyama
Taku
T
Takeda
Shin-Ichi
S
Ichiki
Shota
S
Nakamura
Yuji
Y
Kobayashi
Susumu
S
Uchiyama
Kaoru
K
Nakasone
Shin-Ichi J
SJ
Takayama
Hajime
H
Mita
Yasuhiko
Y
Yamamoto
Yoshihiro
Y
Sambongi
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002151:Calorimetry; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D020539:Sequence Analysis, Protein; D020592:Shewanella / Q000145:classification / Q000201:enzymology*; D013816:Thermodynamics
D001426:Bacterial Proteins; D003574:Cytochrome c Group; D004220:Disulfides; D011994:Recombinant Proteins; C046450:cytochrome C5; D019791:Guanidine; D003545:Cysteine