Proton-Linked Protein Conformational Switching:  Definition of the Alkaline Conformational Transition of Yeast Iso-1-ferricytochrome c

Abstract

The alkaline conformation (state IV) of yeast iso-1-ferricytochrome c and variants in which selected lysyl residues were replaced with alanyl residues has been studied by 1H NMR spectroscopy, electronic spectroscopy, EPR spectroscopy, direct electrochemistry, pH-jump kinetics, and 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. FEBS Lett. 1989, 246, 105−108; Ferrer, J. C.; Guillemette, J. G.; Bogumil, R.; Inglis, S. C.; Smith, M.; Mauk, A. G. J. Am. Chem. Soc. 1993, 115, 7507−7508). The pK a for the conformational equilibrium between state III (native conformation) and state IV of the wild-type protein (8.70(2)) is found to be intermediate between that of the Lys73 bound conformer (8.44(1)) and that of the Lys79 bound conformer (8.82(2)) (0.1 M NaCl, 25 °C) as are the kinetic parameters for the conversion of native protein to each of the two alkaline conformers and the midpoint reduction potentials of the two alkaline forms. The EPR spectra of the Lys73Ala and Lys79Ala variants permit interpretation of the corresponding spectrum of the wild-type protein as the sum of two component conformers. The Lys79Ala 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 the native cytochrome at moderately high pH and appears to undergo a change of axial ligation only under more alkaline conditions (pK a ∼ 10.5). This observation suggests that the coordination of the amine ligands is a significant contribution toward the driving force for formation of the state IV conformers. These results establish the axial ligation of yeast iso-1-ferricytochrome c state IV, characterize the kinetics with which state III converts to state IV, and establish the electrochemical properties and thermal stabilities of the two conformers that constitute state IV. 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 formed, and the relationship of the present results to those reported previously for the formation of state IV from state III.

Nitration of Solvent-exposed Tyrosine 74 on Cytochrome c Triggers Heme Iron-Methionine 80 Bond Disruption

“…As already reported, spectra recorded at different pH values evidenced the transition from the native low spin form (III) at neutral pH to the alkaline low spin form (IV) (Fig. 4B) (13,18,19,24). Several resonances change at high pH (such as those from methyl groups 3, 5, and 8) revealing an overall perturbation of the heme environment.…”

“…The transformation of the state III species (referred to the "neutral" species from now on) into state IV species (the "alkaline" species) upon pH increase is called the alkaline transition (which occurs with a pK a of ϳ9.3) (13, 18 -24). This conformational change is present in all known isoforms of type c cytochrome and implies the displacement of Met-80 from the heme and its substitution by another ligand, Lys-72 or Lys-79 (13,18,24,25), keeping the iron in a low spin state.…”

NMR investigation of ferricytochrome c unfolding: Detection of an equilibrium unfolding intermediate and residual structure in the denatured state

“…Changes in heme substituent shifts for cyt c, therefore, are attributed to replacement of Met-80 with non-native ligands, whereas the His-18 maintains its native orientation. The pattern of heme methyl shifts for species L bears a striking resemblance to that observed for the alkaline form of cyt c, known to have two conformers in which the axial Met ligand is replaced with Lys side chains (Table 1) (33,38). It also bears a strong similarity to the cyanide adducts of Met-80-Ala cyt c (34) and MP-8 (37).…”

“…The Lys residues proposed as candidates for ligation to the heme iron in species L are the same as those proposed to bind heme in the alkaline form of the protein, which forms with a pK of 9 (43). In the alkaline form of Saccharomyces cerevisiae iso-1-cytochrome c, Lys-73 and -79 have been identified as the non-native heme ligands (38). By analogy, Lys-73 and -79 (as well as Lys-72, which is trimethylated in S. cerevisiae cyt c but not in horse cyt c) are candidates for heme ligands in the alkaline form of horse cyt c. Given that the pK for the formation of the alkaline form is substantially lower than that for the side chain of a Lys, it has been proposed that deprotonation of another group (yet to be identified) acts to trigger a conformational change that leads to loss of Met ligation and introduction of the Lys into the heme vicinity.…”

Redox thermodynamics of cytochromes c subjected to urea induced unfolding

“…Wild type recombinant untrimethylated C102T Saccharomyces cerevisiae iso-1-cytochrome c (ycc, hereafter) and the variant Lys72Ala/Lys73Ala/Lys79Ala (KtoA, hereafter) were expressed in E. coli and purified following the procedures described elsewhere [26,27,30], tyrosine replaces the cysteine at position 102 in both species. This substitution prevents dimerization and minimizes autoreduction while resulting in retention of the spectral and the functional properties of the protein [31,32].…”