Hydrophobic effect of trityrosine on heme ligand exchange during folding of cytochrome c.
Effect of a hydrophobic peptide on folding of oxidized cytochrome c (cyt c) is studied with trityrosine. Folding of cyt c was initiated by pH jump from 2.3 (acid-unfolded) to 4.2 (folded). The Soret band of the 2-ms transient absorption spectrum during folding decreased its intensity and red-shifted from 397 to 400 nm by interaction with trityrosine, whereas tyrosinol caused no significant effect. The change in the transient absorption spectrum by interaction with trityrosine was similar to that obtained with 100 mM imidazole, which showed that the population of the intermediate His/His coordinated species increased during folding of cyt c by interaction with trityrosine. The absorption change was biphasic, the fast phase (82+/-9s(-1)) corresponding to the transition from the His/H(2)O to the His/Met coordinated species, whereas the slow phase (24+/-3s(-1)) from His/His to His/Met. By addition of trityrosine, the relative ratio of the slow phase increased, due to increase of the His/His species at the initial stage of folding. According to the resonance Raman spectra of cyt c, the high-spin 6-coordinate and low-spin 6-coordinate species were dominated at pH 2.3 and 4.2, respectively, and these species were not affected by addition of trityrosine. These results demonstrated that the His/His species increased by interaction with trityrosine at the initial stage of cyt c folding, whereas the heme coordination structure was not affected by trityrosine when the protein was completely unfolded or folded. Hydrophobic peptides thus may be useful to study the effects of hydrophobic interactions on protein folding.
Hirota
Shun
S
Department of Physical Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, 607-8414 Kyoto, Japan. hirota@mb.kyoto-phu.ac.jp
Suzuki
Masako
M
Watanabe
Yoshihito
Y
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D045304:Cytochromes c / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D009206:Myocardium / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D017510:Protein Folding; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry*
D000596:Amino Acids; D008024:Ligands; D010455:Peptides; C101095:trityrosine; D014443:Tyrosine; D006418:Heme; D006639:Histidine; D045304:Cytochromes c