PROTON-LINKED PROTEIN CONFORMATIONAL SWITCHING - DEFINITION OF THE ALKALINE CONFORMATIONAL TRANSITION OF YEAST ISO-1-FERRICYTOCHROME-C

The alkaline conformation (state IV) of yeast iso-1-ferricytochrome c and variants in which selected lysyl residues were replaced with alany l residues has been studied by H-1 NMR spectroscopy, electronic spectr oscopy, EPR spectroscopy, direct electrochemistry, pH-jump kinetics, a nd temperature-dependent circular dichroism spectroscopy. On the basis of the NMR studies, Lys73 and Lys79 are shown to replace Met80 as the axial ligand in the two conformers of state IV that were detected in previous studies (Hong, X. L.; Dixon, D. W. FEES Lett. 1989, 246, 105- 108; Ferrer, J. C.; Guillemette, J. G.; Bogumil, R.; Inglis, S. C.; Sm ith, M.; Mauk, A. G. J. Am. Chem. Sec. 1993, 115, 7507-7508). The pK(a ) for the conformational equilibrium between state III (native conform ation) and state N of the wild-type protein (8.70(2)) is found to be i ntermediate between that of the Lys73 bound conformer (8.44(1)) and th at of the Lys79 bound conformer (8.82(2)) (0.1 M NaCl, 25 degrees C) a s are the kinetic parameters for the conversion of native protein to e ach of the two alkaline conformers and the midpoint reduction potentia ls of the two alkaline forms. The EPR spectra of the Lys73Ala and Lys7 9Ala variants permit interpretation of the corresponding spectrum of t he wild-type protein as the sum of two component conformers. The Lys79 Ala variant is slightly more susceptible to thermal denaturation at pH 6.15, but the Lys73Ala variant is less thermally stable than the wild -type cytochrome or the Lys79Ala variant at alkaline pH. The Lys73Ala/ Lys79Ala double variant retains the spectroscopic characteristics of t he native cytochrome at moderately high pH and appears to undergo a ch ange of axial ligation only under more alkaline conditions (pK(a) simi lar to 10.5). This observation suggests that the coordination of the a mine ligands is a significant contribution toward the driving force fo r formation of the state IV conformers. These results establish the ax ial ligation of yeast iso-1-ferricytochrome c state IV, characterize t he kinetics with which state III converts to state IV, and establish t he electrochemical properties and thermal stabilities of the two confo rmers that constitute state TV. The results of this work are discussed with reference to pH-dependent structural behavior of other proteins, the mechanism by which these conformers of the ferricytochrome are fo rmed, and the relationship of the present results to those reported pr eviously for the formation of state IV from state III.