Effect of motional restriction on the unfolding properties of a cytochrome c featuring a His/Met–His/His ligation switch

Abstract

The K72A/K73H/K79A variant of cytochrome c undergoes a reversible change from a His/Met to a His/His axial heme ligation upon urea-induced unfolding slightly below neutral pH. The unfolded form displays a dramatically lower reduction potential than the folded species along with a pseudo-peroxidase activity. We have studied electrochemically the effects of urea-induced unfolding on the protein electrostatically immobilized on an electrode surface functionalized by means of a negatively charged molecular spacer. The latter mimics the electrostatic interaction with the inner mitochondrial membrane. This behavior has been compared with the unfolding of the same species in solution. This system constitutes a model to decipher the role of the above electrostatic interaction in the unfolding of cytochrome c at physiological pH upon interaction with the membrane component phospholipid cardiolipin in the early stages of the apoptosis cascade. We found that immobilization obstacles protein unfolding due to structural constraints at the interface imposed by protein-SAM interaction.

Met80 and Tyr67 affect the chemical unfolding of yeast cytochromec: comparing the solutionvs.immobilized state

“…4 (which would correspond to a 50% population of LS1 and LS2) is about 2.2-2.3 M for both variants. These values are significantly lower than those reported for wt ycc (3.2 M and 3.5 M, at 25 1C and 5 1C, respectively 14,35 ) and its K72A/K73A/K79A and K72A/ K73H/K79A mutants (3.2 M and 3.1 M) at 5 1C, 14,75 indicating that removal of the Fe-Met80 bond favors the unfolding effect of urea. Hence, reduction of the structural constraints that connect the heme center to the polypeptide matrix due to removal of the Fe-Met80 bond lowers the protein resistance to chemical unfolding as found previously for the same immobilized on a MUA-MU SAM.…”

“…Indeed, the E1 0 values of both variants in 6 M urea are similar to those determined for urea unfolded wt ycc and its K72A/K73A/ K79A mutant, 14 which were shown to possess His/His axial coordination. : 33,74,75,103,126,[128][129][130][131][132][133]135,138,139,145,146,148,149 The intrinsic enthalpic contribution depends on the donor properties of the axial heme ligands, the polarity and the electrostatics at the redox center, whereas the intrinsic entropic term is mainly controlled by oxidation-state dependent differences in the conformational degrees of freedom of the polypeptide chain. [74][75][76][77]98,99,103,126,[128][129][130][131][132][133]135,[137][138][139][144][145][146][147][148][149] For both variants, the E1 0 values increase linearly with increasing temperature with and without urea ( Fig.…”

“…1,5,28,30,40,47,73 In vitro, analogous heme axial ligand swapping from His/Met to His/His occurs upon chemical denaturation of both solution 14 and surface-immobilized yeast cytochrome c (ycc). 14,33,34,[74][75][76][77] Under the latter conditions, ycc gains remarkable pseudo-peroxidase activity. 32,34,[75][76][77] Because of their physiological relevance, the conformational equilibria leading to non-native states of cytochrome c have been studied in depth, 1,6,7,14,18,22,[24][25][26][27]41,43,[78][79][80] making it a model system widely used to unravel the mechanistic details of protein folding and unfolding.…”

“…14,33,34,[74][75][76][77] Under the latter conditions, ycc gains remarkable pseudo-peroxidase activity. 32,34,[75][76][77] Because of their physiological relevance, the conformational equilibria leading to non-native states of cytochrome c have been studied in depth, 1,6,7,14,18,22,[24][25][26][27]41,43,[78][79][80] making it a model system widely used to unravel the mechanistic details of protein folding and unfolding. 5,8,9,22,23,25,38,41,78,[81][82][83][84] In this work, we used a combination of spectroscopic and electrochemical techniques to analyze the urea-induced unfolding of the M80A and M80A/Y67A variants of ycc under freely diffusing conditions.…”

Electrocatalytic Properties of Immobilized Heme Proteins: Basic Principles and Applications

“…This can be accomplished only by removing or significantly weakening one of the two axial coordination bonds, either by genetic engineering [41,83,[131][132][133][134][135][136] or as a result of conformational relaxations induced by surface adsorption or denaturing agents such as urea, H 2 O 2 or acidic conditions. Yet, the remarkably efficient coupling to the electrode, due to its structure being optimized to serve as an electron carrier, fulfils one of the major requirements and has pushed the scientific community to search for strategies for endowing cyt c with non-native bioelectrocatalytic properties.…”