Mitochondria are the powerhouses of the cell, whilst their malfunction is related to several human pathologies, including neurodegenerative diseases, cardiovascular diseases, and various types of cancer. In mitochondrial metabolism, cytochrome c is a small soluble heme protein that acts as an essential redox carrier in the respiratory electron transport chain. However, cytochrome c is likewise an essential protein in the cytoplasm acting as an activator of programmed cell death. Such a dual role of cytochrome c in cell life and death is indeed fine-regulated by a wide variety of protein post-translational modifications. In this work, we show how these modifications can alter cytochrome c structure and functionality, thus emerging as a control mechanism of cell metabolism but also as a key element in development and prevention of pathologies.

Post-translational modifications frequently modulate protein functions. Lysine acetylation in particular plays a key role in interactions between respiratory cytochrome c and its metabolic partners. To date, in vivo acetylation of lysines at positions 8 and 53 has specifically been identified in mammalian cytochrome c, but little is known about the structural basis of acetylationinduced functional changes. Here, we independently replaced these two residues in recombinant human cytochrome c with glutamine to mimic lysine acetylation, and then characterized the structure and function of the resulting K8Q and K53Q mutants. We found that the physicochemical features were mostly unchanged in the two acetyl-mimetic mutants, but their thermal stability was significantly altered. Nuclear magnetic resonance chemical shift perturbations of the backbone amide resonances revealed local structural changes, and the thermodynamics and kinetics of electron transfer in mutants immobilized on gold electrodes showed an increase in both protein dynamics and solvent involvement in the redox process. We also observed that the K8Q (but not the K53Q) mutation slightly increased the binding affinity of cytochrome c to its physiological electron donor, cytochrome c 1 -which is a component of mitochondrial complex III, or cytochrome bc 1 -thus suggesting that Lys8 (but not Lys53) is located in the interaction area. Finally, the K8Q and K53Q mutants exhibited reduced efficiency as electron donors to complex IV, or cytochrome c oxidase.

Evaluation of the proton-coupled electron transfer thermodynamics of immobilized hemin is challenging due to the disparity of its electrochemical titration curves reported in the literature. Deviations from the one-electron, one-proton transfer at circumneutral pHs have been commonly ascribed to either the formation of dimeric species or the ionization of a second iron-bound water molecule. Herein, however, we report on non-idealities in the more acidic region, whose onset and extent vary with the nature and concentration of the commonly used phosphate and acetate buffers. It is shown that these deviations originate in the ligand-exchange binding between the oxidized aquo-hemin complex and the anionic components of the buffer, so that they are restricted to the pH interval where these forms coexist. A stepwise approach was developed to quantify unambiguously the apparent and intrinsic binding equilibrium constants. The apparent binding equilibrium constant exhibits a peak-shaped pH dependence, whose maximum is located at approximately the midpoint between the pK a of the iron-bound water and the first pK a of the buffer, and its magnitude is greater for the phosphate than for the acetate buffer. But strikingly, the opposite trend was found for the magnitude of the intrinsic binding equilibrium constants determined from the apparent ones, due to the different relative locations of the phosphoric and acetic pK a values with respect to that of the oxidized aquo-hemin. To probe the role of the heme propionic residues, a similar study was carried out with a propionic-free iron porphyrin containing eight ethyl residues. These substituents decrease the acidity of the iron-bound water, strengthen the iron­(III)–acetate binding, weaken the iron­(III)–dihydrogen phosphate binding, and enable the binding between iron­(III) and monohydrogen phosphate, which was hampered in hemin by the presence of the negatively charged propionate residues. Overall, this work provides a more complete speciation of immobilized iron porphyrins under acidic conditions than previously considered, showing the substitutional lability of the aqua ligand in the oxidized state of the iron center and the reluctance of its hydroxyl counterpart to anion exchange. Knowledge of these redox- and pH-dependent bindings with the buffer components is crucial for a rigorous quantification of the proton-coupled electron transfer and the electrocatalytic activity of iron porphyrins.

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Understanding the molecular basis of the thermal stability and functionality of redox proteins has important practical applications. Here, we show a distinct thermal dependence of the spectroscopic and electrochemical properties of two plastocyanins from the thermophilic cyanobacterium Phormidium laminosum and their mesophilic counterpart from Synechocystis sp. PCC 6803, despite the similarity of their molecular structures. To explore the origin of these differences, we have mimicked the local hydrophobicity in the east patch of the thermophilic protein by replacing a valine of the mesophilic plastocyanin by isoleucine. Interestingly, the resulting mutant approaches the thermal stability, redox thermodynamics, and dynamic coupling of the flexible site motions of the thermophilic protein, indicating the existence of a close connection between the hydrophobic packing of the east patch region of plastocyanin and the functional control and stability of the oxidized and reduced forms of the protein.

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