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The CuA center is a paradigm for the study of long-range biological electron transfer. This metal center is an essential cofactor for terminal oxidases like cytochrome c oxidase, the enzymatic complex responsible for cellular respiration in eukaryotes and in most bacteria. CuA acts as an electron hub by transferring electrons from reduced cytochrome c to the catalytic site of the enzyme where dioxygen reduction takes place. Different electron transfer pathways have been proposed involving a weak axial methionine ligand residue, conserved in all CuA sites. This hypothesis has been challenged by theoretical calculations indicating the lack of electron spin density in this ligand. Here we report an NMR study with selectively labeled methionine in a native CuA. NMR spectroscopy discloses the presence of net electron spin density in the methionine axial ligand in the two alternative ground states of this metal center. Similar spin delocalization observed on two second sphere mutants further supports this evidence. These data provide a novel view of the electronic structure of CuA centers and support previously neglected electron transfer pathways. [ABSTRACT FROM AUTHOR]

Most of the biochemical and biophysical processes of proteins take place at membranes, and are thus under the influence of strong local electric fields, which are likely to affect the structure as well as the reaction mechanism and dynamics. To analyse such electric field effects, biomimetic interfaces may be employed that consist of membrane models deposited on nanostructured metal electrodes. For such devices, surface-enhanced resonance Raman and IR absorption spectroscopy are powerful techniques to disentangle the complex interfacial processes of proteins in terms of rotational diffusion, electron transfer, and protein and cofactor structural changes. The present article reviews the results obtained for the haem protein cytochrome c, which is widely used as a model protein for studying the various reaction steps of interfacial redox processes in general. In addition, it is shown that electric field effects may be functional for the natural redox processes of cytochrome c in the respiratory chain, as well as for the switch from the redox to the peroxidase function, one of the key events preceding apoptosis. The review focuses on the effects of local electric fields on cytochrome c bound to coated electrodes. For such devices that mimic the electrostatic properties of biological membranes surface-sensitive spectroelectrochemicals allow for an in-depth analysis of the molecular processes of the immobilised cytochrome c, contributing to a better understanding of the potential electric-field dependent control of the protein's function. [ABSTRACT FROM AUTHOR]

Examines the proton-coupled electron transfer of cytochrome c (Cyt-c). Attachment of Cyt-c to self-assembled monolayers; Dynamics of electron-transfer reaction of the adsorbed heme protein; Application of time-resolved surfaced enhanced resonance Raman spectroscopy.

Here we report a spectroscopic, electrochemical and computational study of cytochrome c showing that nitration of Tyr74 induces Tyr deprotonation, which is coupled to Met/Lys axial ligand exchange, and results in concomitant gain of peroxidatic activity at physiological pH. [ABSTRACT FROM AUTHOR]

Abstract: In this overview we present recent combined electrochemical, spectroelectrochemical, spectroscopic and computational studies from our group on the electron transfer reactions of cytochrome c and of the primary electron acceptor of cytochrome c oxidase, the CuA site, in biomimetic complexes. Based on these results, we discuss how protein dynamics and thermal fluctuations may impact on protein ET reactions, comment on the possible physiological relevance of these results, and finally propose a regulatory mechanism that may operate in the Cyt/CcO electron transfer reaction in vivo. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference. [Copyright &y& Elsevier]

Abstract: Proteins integrated or transiently bound to membranes are subject to strong electric fields. However, the role of these fields on tuning the reactivity of redox proteins is still an open issue. High electric fields are also present in protein-based electrochemical devices. In this work we report DFT calculations of the electric field effects on ligand binding and on the redox potentials of porphyrin models representing the active sites of typical heme proteins such as cytochrome c, cytochrome c oxidase, cytochrome c peroxidase and cytochrome P450. [Copyright &y& Elsevier]

Abstract Here we investigated the effect of electrostatic interactions and of protein tyrosine nitration of mammalian cytochrome c on the dynamics of the so-called alkaline transition, a pH- and redox-triggered conformational change that implies replacement of the axial ligand Met80 by a Lys residue. Using a combination of electrochemical, time-resolved SERR spectroelectrochemical experiments and molecular dynamics simulations we showed that in all cases the reaction can be described in terms of a two steps minimal reaction mechanism consisting of deprotonation of a triggering group followed by ligand exchange. The p K a a l k values of the transition are strongly modulated by these perturbations, with a drastic downshift upon nitration and an important upshift upon establishing electrostatic interactions with a negatively charged model surface. The value of p K a a l k is determined by the interplay between the acidity of a triggering group and the kinetic constants for the forward and backward ligand exchange processes. Nitration of Tyr74 results in a change of the triggering group from Lys73 in WT Cyt to Tyr74 in the nitrated protein, which dominates the p K a a l k downshift towards physiological values. Electrostatic interactions, on the other hand, result in strong acceleration of the backward ligand exchange reaction, which dominates the p K a a l k upshift. The different physicochemical conditions found here to influence p K a a l k are expected to vary depending on cellular conditions and subcellular localization of the protein, thus determining the existence of alternative conformations of Cyt in vivo. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

Cytochrome c (cyt c) is a small soluble heme protein characterized by a relatively flexible structure, particularly in the ferric form, such that it is able to sample a broad conformational space. Depending on the specific conditions, interactions, and cellular localization, different conformations may be stabilized, which differ in structure, redox properties, binding affinities, and enzymatic activity. The primary function is electron shuttling in oxidative phosphorylation, and is exerted by the so-called native cyt c in the intermembrane mitochondrial space of healthy cells. Under pro-apoptotic conditions, however, cyt c gains cardiolipin peroxidase activity, translocates into the cytosol to engage in the intrinsic apoptotic pathway, and enters the nucleus where it impedes nucleosome assembly. Other reported functions include cytosolic redox sensing and involvement in the mitochondrial oxidative folding machinery. Moreover, post-translational modifications such as nitration, phosphorylation, and sulfoxidation of specific amino acids induce alternative conformations with differential properties, at least in vitro. Similar structural and functional alterations are elicited by biologically significant electric fields and by naturally occurring mutations of human cyt c that, along with mutations at the level of the maturation system, are associated with specific diseases. Here, we summarize current knowledge and recent advances in understanding the different structural, dynamic, and thermodynamic factors that regulate the primary electron transfer function, as well as alternative functions and conformations of cyt c. Finally, we present recent technological applications of this moonlighting protein. [ABSTRACT FROM AUTHOR]

We report a resonance Raman and UV-vis characterization of the active site structure of oxidatively modified forms of cytochrome c (Cyt-c) free in solution and in complexes with cardiolipin (CL). The studied post-translational modifications of Cyt-c include methionine sulfoxidation and tyrosine nitration, which lead to altered heme axial ligation and increased peroxidase activity with respect to those of the wild-type protein. In spite of the structural and activity differences between the protein variants free in solution, binding to CL liposomes induces in all cases the formation of a spectroscopically identical bis-His axial coordination conformer that more efficiently promotes lipid peroxidation. The spectroscopic results indicate that the bis-His form is in equilibrium with small amounts of high-spin species, thus suggesting a labile distal His ligand as the basis for the CL-induced increase in enzymatic activity observed for all protein variants. For Cyt-c nitrated at Tyr74 and sulfoxidized at Met80, the measured apparent binding affinities for CL are ~4 times larger than for wild-type Cyt-c. On the basis of these results, we propose that these post-translational modifications may amplify the pro-apoptotic signal of Cyt-c under oxidative stress conditions at CL concentrations lower than for the unmodified protein. [ABSTRACT FROM AUTHOR]

Nonexponential distance dependenceof the apparent electron-transfer(ET) rate has been reported for a variety of redox proteins immobilizedon biocompatible electrodes, thus posing a physicochemical challengeof possible physiological relevance. We have recently proposed thatthis behavior may arise not only from the structural and dynamicalcomplexity of the redox proteins but also from their interplay withstrong electric fields present in the experimental setups and in vivo(J. Am Chem. Soc.2010, 132, 5769−5778). Therefore, protein dynamics are finely controlledby the energetics of both specific contacts and the interaction betweenthe protein’s dipole moment and the interfacial electric fields.In turn, protein dynamics may govern electron-transfer kinetics throughreorientation from low to high donor–acceptor electronic couplingorientations. Here we present a combined computational and experimentalstudy of WT cytochrome cand the surface mutant K87Cadsorbed on electrodes coated with self-assembled monolayers (SAMs)of varying thickness (i.e., variable strength of the interfacial electricfield). Replacement of the positively charged K87 by a neutral aminoacid allowed us to disentangle protein dynamics and electron tunnelingfrom the reaction kinetics and to rationalize the anomalous distancedependence in terms of (at least) two populations of distinct averageelectronic couplings. Thus, it was possible to recover the exponentialdistance dependence expected from ET theory. These results pave theway for gaining further insight into the parameters that control proteinelectron transfer. [ABSTRACT FROM AUTHOR]

We have employed a combination of protein film voltammetry, time-resolved vibrational spectroelectrochemistry and molecular dynamics simulations to evaluate the electron-transfer reorganization free energy (λ) of cytochrome c (Cyt) in electrostatic complexes that mimic some basic features of protein–protein and protein–lipid interactions. The results reveal the existence of two native-like conformations of Cyt that present significantly different λ values. Conversion from the high to the low λ forms is triggered by electrostatic interactions, and involves the rupture of a weak H-bond between first- (M80) and second-sphere (Y67) ligands of the heme iron, as a distinctive feature of the conformational switch. The two flexible Ω loops operate as transducers of the electrostatic signal. This fine-tuning effect is abolished in the Y67F Cyt mutant, which presents a λ value similar to the WT protein in electrostatic complexes. We propose that interactions of Cyt with the natural redox partner proteins activate a similar mechanism to minimize the reorganization energy of interprotein electron transfer. [ABSTRACT FROM AUTHOR]

Abstract: Time-resolved surface enhanced resonance Raman and surface enhanced infrared absorption spectroscopy have been employed to study the interfacial redox process of cytochrome c (Cyt-c) immobilised on various metal electrodes coated with self-assembled monolayers (SAMs) of carboxyl-terminated mercaptanes. The experiments, carried out with Ag, Au and layered Au–SAM–Ag electrodes, afford apparent heterogeneous electron transfer constants (k relax) that reflect the interplay between electron tunnelling, redox-linked protein structural changes, protein re-orientation, and hydrogen bond re-arrangements in the protein and in the protein/SAM interface. It is shown that the individual processes are affected by the interfacial electric field strength that increases with decreasing thickness of the SAM and increasing difference between the actual potential and the potential of zero-charge. At thick SAMs of mercaptanes including 15 methylene groups, electron tunnelling (kET ) is the rate-limiting step. Pronounced differences for kET and its overpotential-dependence are observed for the three metal electrodes and can be attributed to the different electric-field effects on the free-energy term controlling the tunnelling rate. With decreasing SAM thickness, electron tunnelling increases whereas protein dynamics is slowed down such that for SAMs including less than 10 methylene groups, protein re-orientation becomes rate-limiting, as reflected by the viscosity dependence of k relax. Upon decreasing the SAM thickness from 5 to 1 methylene group, an additional H/D kinetic isotope effect is detected indicating that at very high electric fields re-arrangements of the interfacial or intra-protein hydrogen bond networks limit the rate of the overall redox process. [Copyright &y& Elsevier]

Cytochrome c (Cyt) is a small soluble heme protein with a hexacoordinated heme and functions as an electron shuttle in the mitochondria and in early events of apoptosis when released to the cytoplasm. Using molecular dynamics simulations, we show here that biologically relevant electric fields induce an increased mobility and structural distortion of key protein segments that leads to the detachment of the sixth axial ligand Met80 from the heme iron. This electric-field-induced conformational transition is energetically and entropically driven and leads to a pentacoordinated high spin heme that is characterized by a drastically lowered reduction potential as well as by an increased peroxidase activity. The simulations provide a detailed atomistic picture of the structural effects of the electric field on the structure of Cyt, which allows a sound interpretation of recent experimental results. The observed conformational change may modulate the electron transfer reactions of Cyt in the mitochondria and, furthermore, may constitute a switch from the redox function in the respiratory chain to the peroxidase function in the early events of apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: In this paper we present a combined experimental and theoretical study of the heterogeneous electron transfer reaction of cytochrome c electrostatically adsorbed on metal electrodes coated with monolayers of 6-mercaptohexanoic acid. Molecular dynamics simulations and pathways calculations show that adsorption of the protein leads to a broad distribution of orientations and, thus, to a correspondingly broad distribution of electron transfer rate constants due to the orientation-dependence of the electronic coupling parameter. The adsorbed protein exhibits significant mobility and, therefore, the measured reaction rate is predicted to be a convolution of protein dynamics and tunnelling probabilities for each orientation. This prediction is confirmed by time-resolved surface enhanced resonance Raman which allows for the direct monitoring of protein (re-)orientation and electron transfer of the immobilised cytochrome c. The results provide a consistent explanation for the non-exponential distance-independence of electron transfer rates usually observed for proteins immobilized on electrodes. [Copyright &y& Elsevier]

• Electric fields modulate cytochrome c electron transfer and conformational dynamics. • Kinetic electron transfer and conformational parameters of Cyt c are correlated. • Electric fields may be the switch between canonic and alternative Cyt c functions. • Time-resolved SERR unveils redox-linked conformational changes of Cyt c. • Flexibility perturbation of Cyt c Ω-loops is crucial for its multifunctionality. Cytochrome c (Cyt c) is a multifunctional protein that, in its native conformation, shuttles electrons in the mitochondrial respiratory chain. Conformational transitions that involve replacement of the heme distal ligand lead to the gain of alternative peroxidase activity, which is crucial for membrane permeabilization during apoptosis. Using a time-resolved SERR spectroelectrochemical approach, we found that the key physicochemical parameters that characterize the electron transfer (ET) canonic function and those that determine the transition to alternative conformations are strongly correlated and are modulated by local electric fields (LEF) of biologically meaningful magnitude. The electron shuttling function is optimized at moderate LEFs of around 1 V nm−1. A decrease of the LEF is detrimental for ET as it rises the reorganization energy. Moreover, LEF values below and above the optimal for ET favor alternative conformations with peroxidase activity and downshifted reduction potentials. The underlying proposed mechanism is the LEF modulation of the flexibility of crucial protein segments, which produces a differential effect on the kinetic ET and conformational parameters of Cyt c. These findings might be related to variations in the mitochondrial membrane potential during apoptosis, as the basis for the switch between canonic and alternative functions of Cyt c. Moreover, they highlight the possible role of variable LEFs in determining the function of other moonlighting proteins through modulation of the protein dynamics. [ABSTRACT FROM AUTHOR]

Cytochrome c is a prototypical multifunctional protein that is implicated in a variety of processes that are essential both for sustaining and for terminating cellular life. Typically, alternative functions other than canonical electron transport in the respiratory chain are associated to alternative conformations. In this work we apply a combined experimental and computational study of Cyt c variants to assess whether the parameters that regulate the canonical electron transport function of Cyt c are correlated with those that determine the transition to alternative conformations, using the alkaline transition as a model conformational change. The results show that pK a values of the alkaline transition correlate with the activation energies of the frictionally-controlled electron transfer reaction, and that both parameters are mainly modulated by the flexibility of the Ω-loop 70–85. Reduction potentials and non-adiabatic ET reorganization energies, on the other hand, are both modulated by the flexibilities of the Ω-loops 40–57 and 70–85. Finally, all the measured thermodynamic and kinetic parameters that characterize both types of processes exhibit systematic variations with the dynamics of the hydrogen bond between the axial ligand Met80 and the second sphere ligand Tyr67, thus highlighting the critical role of Tyr67 in controlling canonical and alternative functions of Cyt c. Image 1 [ABSTRACT FROM AUTHOR]