pH dependence of formation of a partially unfolded state of a Lys 73 --> His variant of iso-1-cytochrome c: implications for the alkaline conformational transition of cytochrome c.
The alkaline conformational transition of a lysine 73 --> histidine variant of iso-1-cytochrome c has been studied. The transition has been monitored at 695 nm, a band sensitive to the presence of the heme-methionine 80 bond, at the heme Soret band which is sensitive to the nature of the heme ligand, and by NMR methods. The guanidine hydrochloride dependence of the alkaline conformational transition has also been monitored. The histidine 73 protein has an unusual biphasic alkaline conformational transition at both 695 nm and the heme Soret band, consistent with a three-state process. The conformational transition is fully reversible. An equilibrium model has been developed to account for this behavior. With this model, it has been possible to obtain the acid constant for the trigger group, pK(H), of the low-pH phase from the equilibrium data. A pK(H) value of 6.6 +/- 0.1 in H(2)O was obtained, consistent with a histidine acting as the trigger group. The NMR data for the low-pH phase of the alkaline conformational transition are consistent with an imidazole ligand replacing Met 80. For the high-pH phase of the biphasic alkaline transition, the NMR data are consistent with lysine 79 being the heme ligand. Guanidine hydrochloride m values of 1.67 +/- 0.08 and 1.1 +/- 0.2 kcal mol(-1) M(-1) were obtained for the low- and high-pH phases of the biphasic alkaline transition of the histidine 73 protein, respectively, consistent with a greater structural disruption for the low-pH phase of the transition.
Nelson
C J
CJ
Department of Chemistry and Biochemistry, University of Denver, 2190 East Iliff Avenue, Denver, Colorado 80208-2436, USA.
Bowler
B E
BE
D019943:Amino Acid Substitution / Q000235:genetics*; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics; D019791:Guanidine / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D017186:Titrimetry
D003574:Cytochrome c Group; D007527:Isoenzymes; D006418:Heme; D006639:Histidine; D019791:Guanidine; D008239:Lysine