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Post-translational modifications and naturally occurring mutations of cytochrome c have been recognized as a regulatory mechanism to control its biology. In this work, we investigate the effect of such in vivo chemical modifications of human cytochrome c on its redox properties in the adsorbed state onto an electrode. In particular, tyrosines 48 and 97 have been replaced by the non-canonical amino acid p-carboxymethyl-L-phenylalanine (pCMF), thus mimicking tyrosine phosphorylation. Additionally, tyrosine 48 has been replaced by a histidine producing the natural Y48H pathogenic mutant. Thermodynamics and kinetics of the interfacial electron transfer of wild-type cytochrome c and herein produced variants, adsorbed electrostatically under different local interfacial electric fields, were determined by means of variable temperature cyclic film voltammetry. It is shown that non-native cytochrome c variants immobilized under a low interfacial electric field display redox thermodynamics and kinetics similar to those of wild-type cytochrome c. However, upon increasing the strength of the electric field, the redox thermodynamics and kinetics of the modified proteins markedly differ from those of the wild-type species. The mutations promote stabilization of the oxidized form and a significant increase in the activation enthalpy values that can be ascribed to a subtle distortion of the heme cofactor and/or difference of the amino acid rearrangements rather than to a coarse protein structural change. Overall, these results point to a combined effect of the single point mutations at positions 48 and 97 and the strength of electrostatic binding on the regulatory mechanism of mitochondrial membrane activity, when acting as a redox shuttle protein., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In oxidative phosphorylation, the transfer of electrons from reduced cofactors to molecular oxygen via the electron transport chain (ETC) sustains the electrochemical transmembrane potential needed for ATP synthesis. A key component of the ETC is complex III (CIII, cytochrome bc 1 ), which transfers electrons from reduced ubiquinone to soluble cytochrome c (Cc) coupled to proton translocation into the mitochondrial intermembrane space. One electron from every two donated by hydroquinone at site P is transferred to Cc via the Rieske-cytochrome c 1 (Cc 1 ) pathway. According to recent structural analyses of CIII and its transitory complex with Cc, the interaction between the Rieske subunit and Cc 1 switches intermittently during CIII activity. However, the electrochemical properties of Cc 1 and their function as a wire between Rieske and Cc are rather unexplored. Here, temperature variable cyclic voltammetry provides novel data on the thermodynamics and kinetics of interfacial electron transfer of immobilized Cc 1 . Findings reveal that Cc 1 displays two channels for electron exchange, with a remarkably fast heterogeneous electron transfer rate. Furthermore, the electrochemical properties are strongly modulated by the binding mode of the protein. Additionally, we show that electron transfer from Cc 1 to Cc is thermodynamically favored in the immobilized Cc 1 -Cc complex. Nuclear Magnetic Resonance, HADDOCK, and Surface Plasmon Resonance experiments provide further structural and functional data of the Cc 1 -Cc complex. Our data supports the Rieske-Cc 1 -Cc pathway acting as a unilateral switch thyristor in which redox potential modulation through protein-protein contacts are complemented with the relay-like Rieske behavior., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)