Replacement of a conserved proline and the alkaline conformational change in iso-2-cytochrome c.
Although point mutations usually lead to minor localized changes in protein structure, replacement of conserved Pro-76 with Gly in iso-2-cytochrome c induces a major conformational change. The change in structure results from mutation-induced depression of the pK for transition to an alkaline conformation with altered heme ligation. To assess the importance of position 76 in stabilizing the native versus the alkaline structure, the equilibrium and kinetic properties of the pH-induced conformational change have been compared for normal and mutant iso-2-cytochrome c. The pKapp for the conformational change is reduced from 8.45 (normal iso-2) to 6.71 in the mutant protein (Gly-76 iso-2), suggesting that conservation of Pro-76 may be required to stabilize the native conformation at physiological pH. The kinetics of the conformational change for both the normal and mutant proteins are well-described by a single kinetic phase throughout most of the pH-induced transition zone. Over this pH range, a minimal mechanism proposed for horse cytochrome c [Davis, L. A., Schejter, A., & Hess, G. P. (1974) J. Biol. Chem. 249, 2624-2632] is consistent with the data for normal and mutant yeast iso-2-cytochromes c: NH KH----N + H+ kcf in equilibrium kcb A NH and N are native forms of cytochrome c with a 695-nm absorbance band, A is an alkaline form that lacks the 695-nm band, KH is a proton dissociation constant, and kcf and kcb are microscopic rate constants for the conformational change. The Gly-76 mutation increases kcf by almost 70-fold, but kcb and KH are unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)
Nall
B T
BT
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
Zuniga
E H
EH
White
T B
TB
Wood
L C
LC
Ramdas
L
L
D003574:Cytochrome c Group / Q000235:genetics; D045304:Cytochromes c; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D011392:Proline / Q000235:genetics*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics
D003574:Cytochrome c Group; C032139:iso-2-cytochrome C; D045304:Cytochromes c; D011392:Proline