EPR studies of iso-1-cytochrome c: effect of temperature on two-component spectra of spin label attached to cysteine at positions 102 and 47.
Wild-type iso-1-cytochrome c from Saccharomyces cerevisiae containing naturally occurring cysteine at position 102 and mutated protein S47C (derived from the protein in which C102 had been replaced by threonine) were labeled with cysteine-specific methanethiosulfonate spin label. Continuous wave (CW) electron paramagnetic resonance (EPR) was used to examine the effect of temperature on the behavior of the spin label in the oxidized and reduced forms of wild-type cytochrome c and in the oxidized form of the mutated protein. The computer simulations revealed that the CW EPR spectrum for each form of cytochrome c consists of at least two components [a fast (F) and a slow (S) component], which differ in the values of the rotational correlation times tauRparallel (longitudinal rotational correlation time) and tauRperpendicular (transverse rotational correlation time) and that the relative contributions of the F and S components of the spectra change with temperature. In addition, the values of the rotational correlation times (tauRparallel and tauRperpendicular) for the F component appear to change much more dramatically with the temperature than the respective values for the S component. A large difference between the behavior of the oxidized and reduced wild-type spin-labeled cytochromes c indicates that the temperature-induced unfolding of the protein in the region around C102 progresses more rapidly when cytochrome c is in the oxidized form.
Pyka
J
J
Institute of Molecular Biology, Jagiellonian University, Krakow, Poland.
Osyczka
A
A
Turyna
B
B
Blicharski
W
W
Froncisz
W
W
D003545:Cysteine / Q000737:chemistry*; D003574:Cytochrome c Group / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013053:Spectrophotometry; D013113:Spin Labels; D013696:Temperature
D003574:Cytochrome c Group; D013113:Spin Labels; D003545:Cysteine; D010100:Oxygen