Redox sensitive human mitochondrial aconitase and its interaction with frataxin: In vitro and in silico studies confirm that it takes two to tango.

Mitochondrial aconitase (ACO2) has been postulated as a redox sensor in the tricarboxylic acid cycle. Its high sensitivity towards reactive oxygen and nitrogen species is due to its particularly labile [4Fe-4S]2+ prosthetic group which yields an inactive [3Fe-4S]+ cluster upon oxidation. Moreover, ACO2 was found as a main oxidant target during aging and in pathologies where mitochondrial dysfunction is implied. Herein, we report the expression and characterization of recombinant human ACO2 and its interaction with frataxin (FXN), a protein that participates in the de novo biosynthesis of Fe-S clusters. A high yield of pure ACO2 (≥99%, 22 ± 2 U/mg) was obtained and kinetic parameters for citrate, isocitrate, and cis-aconitate were determined. Superoxide, carbonate radical, peroxynitrite, and hydrogen peroxide reacted with ACO2 with second-order rate constants of 108, 108, 105, and 102 M-1 s-1, respectively. Temperature-induced unfolding assessed by tryptophan fluorescence of ACO2 resulted in apparent melting temperatures of 51.1 ± 0.5 and 43.6 ± 0.2 °C for [4Fe-4S]2+ and [3Fe-4S]+ states of ACO2, sustaining lower thermal stability upon cluster oxidation. Differences in protein dynamics produced by the Fe-S cluster redox state were addressed by molecular dynamics simulations. Reactivation of [3Fe-4S]+-ACO2 by FXN was verified by activation assays and direct iron-dependent interaction was confirmed by protein-protein interaction ELISA and fluorescence spectroscopic assays. Multimer modeling and protein-protein docking predicted an ACO2-FXN complex where the metal ion binding region of FXN approaches the [3Fe-4S]+ cluster, supporting that FXN is a partner for reactivation of ACO2 upon oxidative cluster inactivation.

Correlated electric field modulation of electron transfer parameters and the access to alternative conformations of multifunctional cytochrome c.

Cytochrome c (Cytc) 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 Cytc. 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 Cytc. Moreover, they highlight the possible role of variable LEFs in determining the function of other moonlighting proteins through modulation of the protein dynamics.

Cardiolipin interactions with cytochrome c increase tyrosine nitration yields and site-specificity.

The interaction between cytochrome c and cardiolipin is a relevant process in the mitochondrial redox homeostasis, playing roles in the mechanism of electron transfer to cytochrome c oxidase and also modulating cytochrome c conformation, reactivity and function. Peroxynitrite is a widespread nitrating agent formed in mitochondria under oxidative stress conditions, and can result in the formation of tyrosine nitrated cytochrome c. Some of the nitro-cytochrome c species undergo conformational changes at physiological pH and increase its peroxidase activity. In this work we evaluated the influence of cardiolipin on peroxynitrite-mediated cytochrome c nitration yields and site-specificity. Our results show that cardiolipin enhances cytochrome c nitration by peroxynitrite and targets it to heme-adjacent Tyr67. Cytochrome c nitration also modifies the affinity of protein with cardiolipin. Using a combination of experimental techniques and computer modeling, it is concluded that structural modifications in the Tyr67 region are responsible for the observed changes in protein-derived radical and tyrosine nitration levels, distribution of nitrated proteoforms and affinity to cardiolipin. Increased nitration of cytochrome c in presence of cardiolipin within mitochondria and the gain of peroxidatic activity could then impact events such as the onset of apoptosis and other processes related to the disruption of mitochondrial redox homeostasis.

Electron transfer and conformational transitions of cytochrome c are modulated by the same dynamical features.

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 pKa 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.

Aconitases: Non-redox Iron-Sulfur Proteins Sensitive to Reactive Species.

Mammalian aconitases (mitochondrial and cytosolic isoenzymes) are unique iron-sulfur cluster-containing proteins in which the metallic center participates in the catalysis of a non-redox reaction. Within the cubane iron-sulfur cluster of aconitases only three of the four iron ions have cysteine thiolate ligands; the fourth iron ion (Feα) is solvent exposed within the active-site pocket and bound to oxygen atoms from either water or substrates to be dehydrated. The catalyzed reaction is the reversible isomerization of citrate to isocitrate with an intermediate metabolite, cis-aconitate. The cytosolic isoform of aconitase is a moonlighting enzyme; when intracellular iron is scarce, the complete disassembly of the iron-sulfur cluster occurs and apo-aconitase acquires the function of an iron responsive protein and regulates the translation of proteins involved in iron metabolism. In the late 1980s and during the 1990s, cumulative experimental evidence pointed out that aconitases are main targets of reactive oxygen and nitrogen species such as superoxide radical (O2•-), hydrogen peroxide (H2O2), nitric oxide (•NO), and peroxynitrite (ONOO-). These intermediates are capable of oxidizing the cluster, which leads to iron release and consequent loss of the catalytic activity of aconitase. As the reaction of the Fe-S cluster with O2•- is fast (∼107 M-1 s-1), quite specific, and reversible in vivo, quantification of active aconitase has been used to evaluate O2•- formation in cells. While •NO is modestly reactive with aconitase, its reaction with O2•- yields ONOO-, a strong oxidant that readily leads to the disruption of the Fe-S cluster. In the case of cytosolic aconitase, it has been seen that H2O2 and •NO promote activation of iron responsive protein activity in cells. Proteomic advances in the 2000s confirmed that aconitases are main targets of reactive species in cellular models and in vivo, and other post-translational oxidative modifications such as protein nitration and carbonylation have been detected. Herein, we (1) outline the particular structural features of aconitase that make these proteins specific targets of reactive species, (2) characterize the reactions of O2•-, H2O2, •NO, and ONOO- and related species with aconitases, (3) discuss how different oxidative post-translational modifications of aconitase impact the different functions of aconitases, and (4) argue how these proteins might function as redox sensors within different cellular compartments, regulating citrate concentration and efflux from mitochondria, iron availability in the cytosol, and cellular oxidant production.

The alkaline transition of cytochrome c revisited: Effects of electrostatic interactions and tyrosine nitration on the reaction dynamics.

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 pKaalk 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 pKaalk 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 pKaalk downshift towards physiological values. Electrostatic interactions, on the other hand, result in strong acceleration of the backward ligand exchange reaction, which dominates the pKaalk upshift. The different physicochemical conditions found here to influence pKaalk 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.

Rapid peroxynitrite reduction by human peroxiredoxin 3: Implications for the fate of oxidants in mitochondria.

Mitochondria are main sites of peroxynitrite formation. While at low concentrations mitochondrial peroxynitrite has been associated with redox signaling actions, increased levels can disrupt mitochondrial homeostasis and lead to pathology. Peroxiredoxin 3 is exclusively located in mitochondria, where it has been previously shown to play a major role in hydrogen peroxide reduction. In turn, reduction of peroxynitrite by peroxiredoxin 3 has been inferred from its protective actions against tyrosine nitration and neurotoxicity in animal models, but was not experimentally addressed so far. Herein, we demonstrate the human peroxiredoxin 3 reduces peroxynitrite with a rate constant of 1 × 107 M-1 s-1 at pH 7.8 and 25 °C. Reaction with hydroperoxides caused biphasic changes in the intrinsic fluorescence of peroxiredoxin 3: the first phase corresponded to the peroxidatic cysteine oxidation to sulfenic acid. Peroxynitrite in excess led to peroxiredoxin 3 hyperoxidation and tyrosine nitration, oxidative post-translational modifications that had been previously identified in vivo. A significant fraction of the oxidant is expected to react with CO2 and generate secondary radicals, which participate in further oxidation and nitration reactions, particularly under metabolic conditions of active oxidative decarboxylations or increased hydroperoxide formation. Our results indicate that both peroxiredoxin 3 and 5 should be regarded as main targets for peroxynitrite in mitochondria.

Multifunctional Cytochrome c: Learning New Tricks from an Old Dog.

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.

Alternative Conformations of Cytochrome c: Structure, Function, and Detection.

Cytochrome c (cyt c) is a cationic hemoprotein of ∼100 amino acid residues that exhibits exceptional functional versatility. While its primary function is electron transfer in the respiratory chain, cyt c is also recognized as a key component of the intrinsic apoptotic pathway, the mitochondrial oxidative protein folding machinery, and presumably as a redox sensor in the cytosol, along with other reported functions. Transition to alternative conformations and gain-of-peroxidase activity are thought to further enable the multiple functions of cyt c and its translocation across cellular compartments. In vitro, direct interactions of cyt c with cardiolipin, post-translational modifications such as tyrosine nitration, phosphorylation, methionine sulfoxidation, mutations, and even fine changes in electrical fields lead to a variety of conformational states that may be of biological relevance. The identification of these alternative conformations and the elucidation of their functions in vivo continue to be a major challenge. Here, we unify the knowledge of the structural flexibility of cyt c that supports functional moonlighting and review biochemical and immunochemical evidence confirming that cyt c undergoes conformational changes during normal and altered cellular homeostasis.

Active Site Structure and Peroxidase Activity of Oxidatively Modified Cytochrome c Species in Complexes with Cardiolipin.

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.

Coupling of tyrosine deprotonation and axial ligand exchange in nitrocytochrome c.

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.

Electrostatically driven second-sphere ligand switch between high and low reorganization energy forms of native cytochrome c.

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.

Topography of tyrosine residues and their involvement in peroxidation of polyunsaturated cardiolipin in cytochrome c/cardiolipin peroxidase complexes.

Formation of cytochrome c (cyt c)/cardiolipin (CL) peroxidase complex selective toward peroxidation of polyunsaturated CLs is a pre-requisite for mitochondrial membrane permeabilization. Tyrosine residues - via the generation of tyrosyl radicals (Tyr) - are likely reactive intermediates of the peroxidase cycle leading to CL peroxidation. We used mutants of horse heart cyt c in which each of the four Tyr residues was substituted for Phe and assessed their contribution to the peroxidase catalysis. Tyr67Phe mutation was associated with a partial loss of the oxygenase function of the cyt c/CL complex and the lowest concentration of H(2)O(2)-induced Tyr radicals in electron paramagnetic resonance (EPR) spectra. Our MS experiments directly demonstrated decreased production of CL-hydroperoxides (CL-OOH) by Tyr67Phe mutant. Similarly, oxidation of a phenolic substrate, Amplex Red, was affected to a greater extent in Tyr67Phe than in three other mutants. Tyr67Phe mutant exerted high resistance to H(2)O(2)-induced oligomerization. Measurements of Tyr fluorescence, hetero-nuclear magnetic resonance (NMR) and computer simulations position Tyr67 in close proximity to the porphyrin ring heme iron and one of the two axial heme-iron ligand residues, Met80. Thus, the highly conserved Tyr67 is a likely electron-donor (radical acceptor) in the oxygenase half-reaction of the cyt c/CL peroxidase complex.

Mitochondrial protein tyrosine nitration.

Mitochondria are primary loci for the intracellular formation and reactions of reactive oxygen and nitrogen species including superoxide (O2•⁻), hydrogen peroxide (H2O2) and peroxynitrite (ONOO⁻). Depending on formation rates and steady-state levels, the mitochondrial-derived short-lived reactive species contribute to signalling events and/or mitochondrial dysfunction through oxidation reactions. Among relevant oxidative modifications in mitochondria, the nitration of the amino acid tyrosine to 3-nitrotyrosine has been recognized in vitro and in vivo. This post-translational modification in mitochondria is promoted by peroxynitrite and other nitrating species and can disturb organelle homeostasis. This study assesses the biochemical mechanisms of protein tyrosine nitration within mitochondria, the main nitration protein targets and the impact of 3-nitrotyrosine formation in the structure, function and fate of modified mitochondrial proteins. Finally, the inhibition of mitochondrial protein tyrosine nitration by endogenous and mitochondrial-targeted antioxidants and their physiological or pharmacological relevance to preserve mitochondrial functions is analysed.

pH-sensitive binding of cytochrome c to the inner mitochondrial membrane. Implications for the participation of the protein in cell respiration and apoptosis.

Cytochrome c exhibits two positively charged sites: site A containing lysine residues with high pKa values and site L containing ionizable groups with pKaobs values around 7.0. This protein feature implies that cytochrome c can participate in the fusion of mitochondria and have its detachment from the inner membrane regulated by cell acidosis and alkalosis. In this study, we demonstrated that both horse and tuna cytochrome c exhibited two types of binding to inner mitochondrial membranes that contributed to respiration: a high-affinity and low-efficiency pH-independent binding (microscopic dissociation constant Ksapp2, approximately 10 nM) and a low-affinity and high-efficiency pH-dependent binding that for horse cytochrome c had a pKa of approximately 6.7. For tuna cytochrome c (Lys22 and His33 replaced with Asn and Trp, respectively), the effect of pH on Ksapp1 was less striking than for the horse heme protein, and both tuna and horse cytochrome c had closed Ksapp1 values at pH 7.2 and 6.2, respectively. Recombinant mutated cytochrome c H26N and H33N also restored the respiration of the cytochrome c-depleted mitoplast in a pH-dependent manner. Consistently, the detachment of cytochrome c from nondepleted mitoplasts was favored by alkalinization, suggesting that site L ionization influences the participation of cytochrome c in the respiratory chain and apoptosis.

Disruption of the M80-Fe ligation stimulates the translocation of cytochrome c to the cytoplasm and nucleus in nonapoptotic cells.

Native cytochrome c (cyt c) has a compact tertiary structure with a hexacoordinated heme iron and functions in electron transport in mitochondria and apoptosis in the cytoplasm. However, the possibility that protein modifications confer additional functions to cyt c has not been explored. Disruption of methionine 80 (M80)-Fe ligation of cyt c under nitrative stress has been reported. To model this alteration and determine if it confers new properties to cyt c, a cyt c mutant (M80A) was constitutively expressed in cells. M80A-cyt c has increased peroxidase activity and is spontaneously released from mitochondria, translocating to the cytoplasm and nucleus in the absence of apoptosis. Moreover, M80A models endogenously nitrated cyt c because nitration of WT-cyt c is associated with its translocation to the cytoplasm and nucleus. Further, M80A cyt c may up-regulate protective responses to nitrative stress. Our findings raise the possibility that endogenous protein modifications that disrupt the M80-Fe ligation (such as tyrosine nitration) stimulate nuclear translocation and confer new functions to cyt c in nonapoptotic cells.

Nitration of solvent-exposed tyrosine 74 on cytochrome c triggers heme iron-methionine 80 bond disruption. Nuclear magnetic resonance and optical spectroscopy studies.

Cytochrome c, a mitochondrial electron transfer protein containing a hexacoordinated heme, is involved in other physiologically relevant events, such as the triggering of apoptosis, and the activation of a peroxidatic activity. The latter occurs secondary to interactions with cardiolipin and/or post-translational modifications, including tyrosine nitration by peroxynitrite and other nitric oxide-derived oxidants. The gain of peroxidatic activity in nitrated cytochrome c has been related to a heme site transition in the physiological pH region, which normally occurs at alkaline pH in the native protein. Herein, we report a spectroscopic characterization of two nitrated variants of horse heart cytochrome c by using optical spectroscopy studies and NMR. Highly pure nitrated cytochrome c species modified at solvent-exposed Tyr-74 or Tyr-97 were generated after treatment with a flux of peroxynitrite, separated, purified by preparative high pressure liquid chromatography, and characterized by mass spectrometry-based peptide mapping. It is shown that nitration of Tyr-74 elicits an early alkaline transition with a pKa = 7.2, resulting in the displacement of the sixth and axial iron ligand Met-80 and replacement by a weaker Lys ligand to yield an alternative low spin conformation. Based on the study of site-specific Tyr to Phe mutants in the four conserved Tyr residues, we also show that this transition is not due to deprotonation of nitro-Tyr-74, but instead we propose a destabilizing steric effect of the nitro group in the mobile Omega-loop of cytochrome c, which is transmitted to the iron center via the nearby Tyr-67. The key role of Tyr-67 in promoting the transition through interactions with Met-80 was further substantiated in the Y67F mutant. These results therefore provide new insights into how a remote post-translational modification in cytochrome c such as tyrosine nitration triggers profound structural changes in the heme ligation and microenvironment and impacts in protein function.

Mitochondrial aconitase reaction with nitric oxide, S-nitrosoglutathione, and peroxynitrite: mechanisms and relative contributions to aconitase inactivation.

Using highly purified recombinant mitochondrial aconitase, we determined the kinetics and mechanisms of inactivation mediated by nitric oxide (*NO), nitrosoglutathione (GSNO), and peroxynitrite (ONOO(-)). High *NO concentrations are required to inhibit resting aconitase. Brief *NO exposures led to a reversible inhibition competitive with isocitrate (K(I)=35 microM). Subsequently, an irreversible inactivation (0.65 M(-1) s(-1)) was observed. Irreversible inactivation was mediated by GSNO also, both in the absence and in the presence of substrates (0.23 M(-1) s(-1)). Peroxynitrite reacted with the [4Fe-4S] cluster, yielding the inactive [3Fe-4S] enzyme (1.1 x 10(5) M(-1) s(-1)). Carbon dioxide enhanced ONOO(-)-dependent inactivation via reaction of CO(3)*(-) with the [4Fe-4S] cluster (3 x 10(8) M(-1) s(-1)). Peroxynitrite also induced m-aconitase tyrosine nitration but this reaction did not contribute to enzyme inactivation. Computational modeling of aconitase inactivation by O(2)*(-) and *NO revealed that, when NO is produced and readily consumed, measuring the amount of active aconitase remains a sensitive method to detect variations in O(2)*(-) production in cells but, when cells are exposed to high concentrations of NO, aconitase inactivation does not exclusively reflect changes in rates of O(2)*(-) production. In the latter case, extents of aconitase inactivation reflect the formation of secondary reactive species, specifically ONOO(-) and CO(3)*(-), which also mediate m-aconitase tyrosine nitration, a footprint of reactive *NO-derived species.