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.
The K72A/K73H/K79A mutant of yeast iso-1-cytochrome c immobilized on a conductive substrate reversibly interconverts between the native-like, His-Met heme-ligated form and a His-His-ligated conformer with remarkably different redox and enzymatic properties. This transition is activated by changing the pH in a narrow range around neutrality
The voltage-dependent anion channel 1 (VDAC-1) is an important protein of the outer mitochondrial membrane that transports energy metabolites and is involved in apoptosis. The available structures of VDAC proteins show a wide β-stranded barrel pore, with its N-terminal α-helix (N-α) bound to its interior. Electrophysiology experiments revealed that voltage, its polarity, and membrane composition modulate VDAC currents. Experiments with VDAC-1 mutants identified amino acids that regulate the gating process. However, the mechanisms for how these factors regulate VDAC-1, and which changes they trigger in the channel, are still unknown. In this study, molecular dynamics simulations and single-channel experiments of VDAC-1 show agreement for the current-voltage relationships of an "open" channel and they also show several subconducting transient states that are more cation selective in the simulations. We observed voltage-dependent asymmetric distortions of the VDAC-1 barrel and the displacement of particular charged amino acids. We constructed conformational models of the protein voltage response and the pore changes that consistently explain the protein conformations observed at opposite voltage polarities, either in phosphatidylethanolamine or phosphatidylcholine membranes. The submicrosecond VDAC-1 voltage response shows intrinsic structural changes that explain the role of key gating amino acids and support some of the current gating hypotheses. These voltage-dependent protein changes include asymmetric barrel distortion, its interaction with the membrane, and significant displacement of N-α amino acids.
Understanding the factors governing the rate of electron transfer processes in proteins is crucial not only to a deeper understanding of redox processes in living organisms but also for the design of efficient devices featuring biological molecules. Here, molecular dynamics simulations performed on native azurin and four chimeric cupredoxins allow for the calculation of the reorganization energy and of structure-related quantities that were used to clarify the molecular determinants to the dynamics/function relationship in blue copper proteins. We find that the dynamics of the small, metal-binding loop region controls the outer-sphere reorganization energy not only by determining the exposure of the active site to solvent but also through the modulation of the redox-dependent rearrangement of the whole protein scaffold and of the surrounding water molecules.
Perturbed matrix method calculations are performed on a diheme cytochrome c (DHC) protein, in order to assign previously experimentally detemined reduction potentials (E) to their corresponding heme groups. Very good agreement between calculated values to experimental data prove that the present approach can be used as a predictive tool of redox thermodynamic properties of multicenter redox proteins in the absence of experimental data, or in synergy with state-of-the art spectroscopic and electrochemical approaches to obtain a detailed picture of electron transfer processes within these complex systems.
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