Resolving the individual components of a pH-induced conformational change.
This communication introduces a simple method to determine the pKs of microscopic ionizations from complex titration curves. We used this approach to study the alkaline transition (pH-dependent ligand exchange) of mitochondrial cytochrome c. The linearization of titration curves permitted resolution of two to three limiting microscopic ionizations. By combining these data with studies of the temperature dependence of ligand-exchange equilibria, we found evidence that the alkaline transition comprises two chemically distinct processes: the deprotonation of the alternative ligands and the break of the iron-methionine ligation bond. We also noted that, in the horse and untrimethylated S. cerevisiae iso-1 cytochromes c, the permissible deprotonation of the epsilon-amino group of Lys(72) allows formation of an alkaline isomer at lower pH, with lesser stability, which leads to hysteresis in the titration curves. The linearization of the titration curves for different cytochromes c thus brings insight on the microscopic contributions to conformational stability.
Blouin
C
C
Department of Biochemistry and Molecular Biology, Dalhousie University, Nova Scotia B3H 4H7, Canada.
Guillemette
J G
JG
Wallace
C J
CJ
D000818:Animals; D001120:Arginine / Q000378:metabolism; D012809:Colon, Sigmoid; D003574:Cytochrome c Group / Q000378:metabolism*; D045304:Cytochromes c; D006736:Horses / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007536:Isomerism; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017186:Titrimetry
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D029701:Saccharomyces cerevisiae Proteins; D045304:Cytochromes c; D001120:Arginine; D008715:Methionine; D007501:Iron; D008239:Lysine