Roles of a short connecting disulfide bond in the stability and function of psychrophilic Shewanella violacea cytochrome c 5*

Abstract

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.

Correlation between the Stability and Redox Potential of Three Homologous Cytochromescfrom Two Thermophiles and One Mesophile

“…19) It is likely that the somewhat conserved environment of mitochondria in eukaryotic cells can maintain the conserved cyt c interior around the heme. For further examples in an expanded range of growth temperatures, we are currently examining class IE cyts c isolated from mesophilic Shewanella amazonensis and psychrophilic Shewanella violacea, 20) which grow optimally at 35 and 8 C respectively.…”

The Respiratory System of the PiezophilePhotobacterium profundumSS9 Grown under Various Pressures

“…Many y To whom correspondence should be addressed. Fax: +81-3-5317-9433; E-mail: htamegai@chs.nihon-u.ac.jp studies have been carried out on the respiratory system of S. violacea and on related strains for comparison, [19][20][21][22] but it remains unknown whether this phenomenon observed in S. violacea is universal to piezophiles.…”

Heme Is Not Required forAquifex aeolicusCytochromec555Polypeptide Folding

“…Thermal denaturation experiments were carried out by monitoring CD spectra in a pressure-proof cell compartment (JASCO) that was attached to a JASCO J-820 CD spectrometer. 14) Protein solutions of the air-oxidized holo AA c 555 and holo and apo C12A/C15A variants (20 mM) in 20 mM sodium acetate (pH 5.0) were subjected to analysis. The temperature-dependent CD ellipticity change at 222 nm was monitored in a cuvette of 1 mm pathlength.…”