Horse Heart Cytochrome Research Articles

Ferrous nitrosylated cytochrome c: The unusual strength of the proximal His18-Fe bond

NO binding to horse heart cytochrome c (hhcyt c) has been investigated as a function of pH by both optical absorption and EPR spectroscopies. Lowering pH from 3.5 to 1.5 induces: (i) a blue-shift of the maximum of the optical absorption spectrum in the Soret region from 415 to about 404 nm, and (ii) the appearance of a strong three hyperfine splitting in the gz region of the EPR spectrum. Both spectroscopic features indicate the cleavage of the proximal His18-Fe(II)-NO bond giving rise to the five-coordinated Fe(II)-NO species. By quantification of the relative weight for the six- and the five-coordinated component in the EPR spectra, the pKa value was determined. The apparent pKa of the proximal His Nε atom (1.8 ± 0.1) is unusually low for a ferrous nitrosylated form since in all investigated ferrous NO-bound heme-proteins the pKa value for the cleavage of the proximal His-Fe(II) bond ranges between 3.7 and 5.8. The pKa value of ferrous nitrosylated hhcyt c indicates that the strength of the proximal His18-Fe(II) bond (= 27.9 kJ/mol) is about 10–22 kJ/mol higher than that observed in all investigated heme-proteins. The strong coordination of the heme-Fe atom by His18 is extremely important to maintain the redox efficiency of cyt c and to keep apoptosis under control. This is a crucial point in tissues, such as retina, where apoptosis might trigger macular degenerative processes.

The Role of Electrostatic Binding Interfaces in the Performance of Bacterial Reaction Center Biophotoelectrodes.

Photosynthetic reaction centers (RCs) efficiently capture and convert solar radiation into electrochemical energy. Accordingly, RCs have the potential as components in biophotovoltaics, biofuel cells, and biosensors. Recent biophotoelectrodes containing the RC from the bacterium Rhodobacter sphaeroides utilize a natural electron donor, horse heart cytochrome c (cyt c), as an electron transfer mediator with the electrode. In this system, electrostatic interfaces largely control the protein-electrode and protein-protein interactions necessary for electron transfer. However, recent studies have revealed kinetic bottlenecks in cyt-mediated electron transfer that limit biohybrid photoelectrode efficiency. Here, we seek to understand how changing protein-protein and protein-electrode interactions influence RC turnover and biophotoelectrode efficiency. The RC-cyt c binding interaction was modified by substituting interfacial RC amino acids. Substitutions Asn-M188 to Asp and Gln-L264 to Glu, which are known to produce a higher cyt-binding affinity, led to a decrease in the RC turnover frequency (TOF) at the electrode, suggesting that a decrease in cyt c dissociation was rate-limiting in these RC variants. Conversely, an Asp-M88 to Lys substitution producing a lower binding affinity had little effect on the RC TOF, suggesting that a decrease in the cyt c association rate was not a rate-limiting factor. Modulating the electrode surface with a self-assembled monolayer that oriented the cyt c to face the electrode did not affect the RC TOF, suggesting that the orientation of cyt c was also not a rate-limiting factor. Changing the ionic strength of the electrolyte solution had the most potent impact on the RC TOF, indicating that cyt c mobility was important for effective electron donation to the photo-oxidized RC. An ultimate limitation for the RC TOF was that cyt c desorbed from the electrode at ionic strengths above 120 mM, diluting its local concentration near the electrode-adsorbed RCs and resulting in poor biophotoelectrode performance. These findings will guide further tuning of these interfaces for improved performance.

Tripeptide Self-Assembled Monolayers as Biocompatible Surfaces for Cytochrome c Electrochemistry.

Biocompatible tripeptide self-assembled monolayers (SAMs) are designed with a carboxylate group on the terminal amino acid (glutamate, aspartate, or amino adipate) to electrostatically attract the lysine groups around the heme crevice in horse heart cytochrome c (cyt c), creating an electroactive protein/tripeptide/Au interfacial structure. Exposing the peptide/Au electrode to cyt c resulted in an 11 ± 3 pmol/cm2 electroactive protein surface coverage. Topographical images of the interfacial structure are obtained down to single-protein resolution by atomic force microscopy. Uniform protein monolayer assemblies are formed on the Au electrode with no major surface roughness changes. The cyt c/peptide/Au electrode systems were examined electrochemically to probe surface charge effects on the redox thermodynamics and kinetics of cyt c. Neutralization of protein surface charge due to adsorption on anionic COOH-terminated SAMs was found to change the formal potential, as determined by cyclic voltammetry. The cyt c/peptide/Au electrodes exhibit formal potentials shifted to more positive values, have a surface carboxylic acid pKa of 6 or higher, and produce effective cyt c surface charges (Zox) of -6 to -14. The Marcus theory is utilized to determine the protein electron transfer rates, which are ∼5 times faster for cyt c/tripeptide/Au compared to cyt c/11-mercaptoundecanoic acid SAMs of similar chain lengths.

Modeling the molecular dynamics of cytochrome C in aqueous and water–methanol environment

One-microsecond molecular dynamics of horse heart cytochrome C was modeled in aqueous and water–methanol environment. It was shown that the coordination bond between Met-80 sulfur and heme iron is broken in water– methanol solution.

New insights into the folding-unfolding mechanism and conformations of cytochrome C.

Metalloproteins account for over one-third of all proteins in nature and play important roles in biological processes. The formation of the native structures of metalloproteins requires not only the correct folding of the polypeptide chains but also the proper incorporation of metal cofactors. Understanding the folding mechanism of metalloproteins has been challenging. Horse heart cytochrome C (cytc) is a classical model system for protein folding studies. Although a large number of ensemble studies have been carried out to characterize the folding mechanism of cytc, there is still a significant debate on the folding mechanism and the existence of the proposed “foldons”. Here, we used single-molecule optical tweezers to probe the mechanical folding–unfolding behaviors of cytc at the single-molecule level. By directly monitoring the folding and unfolding of holo-cytc, we revealed novel insights into the folding of cytc. Our results showed that the structural elements that are distant from the N- and C-termini can exist as a short-lived intermediate, a finding that contrasts with the general belief that the folding and packing of the N- and C-terminal helices are prerequisites for the folding of other structural elements in cytc. In addition, our results present strong evidence that apo-cytc, which has been long believed to be a random coil, is not a true random coil, and weak interactions within the unfolded polypeptide chain exist. Our results bring new insights into our understanding of the folding mechanisms of heme proteins as well as the role of heme in the folding process.

Kinetics and Energetics of Intramolecular Electron Transfer in Single-Point Labeled TUPS-Cytochrome c Derivatives.

Electron transfer within and between proteins is a fundamental biological phenomenon, in which efficiency depends on several physical parameters. We have engineered a number of horse heart cytochrome c single-point mutants with cysteine substitutions at various positions of the protein surface. To these cysteines, as well as to several native lysine side chains, the photoinduced redox label 8-thiouredopyrene-1,3,6-trisulfonate (TUPS) was covalently attached. The long-lived, low potential triplet excited state of TUPS, generated with high quantum efficiency, serves as an electron donor to the oxidized heme c. The rates of the forward (from the label to the heme) and the reverse (from the reduced heme back to the oxidized label) electron transfer reactions were obtained from multichannel and single wavelength flash photolysis absorption kinetic experiments. The electronic coupling term and the reorganization energy for electron transfer in this system were estimated from temperature-dependent experiments and compared with calculated parameters using the crystal and the solution NMR structure of the protein. These results together with the observation of multiexponential kinetics strongly support earlier conclusions that the flexible arm connecting TUPS to the protein allows several shortcut routes for the electron involving through space jumps between the label and the protein surface.

Reactions with Proteins of Three Novel Anticancer Platinum(II) Complexes Bearing N-Heterocyclic Ligands.

Three novel platinum(II) complexes bearing N-heterocyclic ligands, i.e., Pt2c, Pt-IV and Pt-VIII, were previously prepared and characterized. They manifested promising in vitro anticancer properties associated with non-conventional modes of action. To gain further mechanistic insight, we have explored here the reactions of these Pt compounds with a few model proteins, i.e., hen egg white lysozyme (HEWL), bovine pancreatic ribonuclease (RNase A), horse heart cytochrome c (Cyt-c) and human serum albumin (HSA), primarily through ESI MS analysis. Characteristic and variegate patterns of reactivity were highlighted in the various cases that appear to depend both on the nature of the Pt complex and of the interacting protein. The protein-bound Pt fragments were identified. In the case of the complex Pt2c, the adducts formed upon reaction with HEWL and RNase A were further characterized by solving the respective crystal structures: this allowed us to determine the exact location of the various Pt binding sites. The implications of the obtained results are discussed in relation to the possible mechanisms of action of these innovative anticancer Pt complexes.

Effect of confinement of horse heart cytochrome c and formate dehydrogenase from Candida boidinii on mesoporous carbons on their catalytic activity

This study reports the immobilization of two biocatalysts (e.g., cytochrome c—Cyt c—and the non-metalloenzyme formate dehydrogenase from Candida boidinii–cbFDH) on a series of mesoporous carbons with controlled pore sizes. The catalytic activity of the nanoconfined proteins was correlated with the pore size distribution of the carbon materials used as supports. The electrochemical behaviour of nanoconfined Cyt c showed direct electron transfer electroactivity in pore sizes matching tightly the protein dimension. The pseudo-peroxidase activity towards H2O2 reduction was enhanced at pH 4.0, due to the protein conformational changes. For cbFDH, the reduction of CO2 towards formic acid was evaluated for the nanoconfined protein, in the presence of nicotinamide adenine dinucleotide (NADH). The carbons displayed different cbFDH uptake capacity, governed by the dimensions of the main mesopore cavities and their accessibility through narrow pore necks. The catalytic activity of nanoconfined cbFDH was largely improved, compared to its performance in free solution. Regardless of the carbon support used, the production of formic acid was higher upon immobilization with lower nominal cbFDH:NADH ratios.

Lysine 72 substitutions differently affect lipid membrane permeabilizing and proapoptotic activities of horse heart cytochrome c

Peroxidase activity of cytochrome c (cyt c)/cardiolipin (CL) complex is supposed to be involved in the initiation of apoptosis via peroxidative induction of mitochondrial membrane permeabilization. As cyt c binding to CL-containing membranes is at least partially associated with electrostatic protein/lipid interaction, we screened single-point mutants of horse heart cyt c with various substitutions of lysine at position 72, considered to play a significant role in both the binding and peroxidase activity of the protein. Contrary to expectations, K72A, K72R and K72L substitutions exerted slight effects on both the cyt c binding to CL-containing liposomal membranes and the cyt c/H2O2-induced calcein leakage from liposomes, used here as a membrane permeabilization assay. Both the binding and permeabilization were decreased to various extents, but not significantly, in the case of K72E and K72N mutants. A drastic difference was found between the sequence of the permeabilizing activities of the cyt c variants and the previously described order of their proapoptotic activities (Chertkova et al., 2008).

Destabilization effect of imidazolium cation-Hofmeister anion salts on cytochrome c

We have analyzed an effect of imidazolium cation-Hofmeister anion salts on stability of basic horse heart cytochrome c (cyt c) at pH4.5 (net charge +17). The effect of salts consisting of imidazolium cations, 1-ethyl-3-methylimidazolium (EMIm+) and 1-butyl-3-methylimidazolium (BMIm+), and five anions: chloride, bromide, iodide, nitrate, and thiocyanate on thermal and pH stability of cyt c was compared with the effect of corresponding sodium salts. Correlation between parameter of dTtrs/d [ion] (Ttrs; thermal midpoints) with surface tension changes of solvent in the presence of both imidazolium and sodium salts implies direct interaction between ions and proteins. Surprisingly, the imidazolium salts have more pronounced destabilization effect on highly positively charged cyt c than the corresponding sodium counterparts. Our analysis suggests the direct interaction of imidazolium cations with polypeptide chain, in analogy to guanidium cation, but the destabilization effect is significantly strengthened by decreased surface tension of imidazolium salt solvents. Comparison of an effect of imidazolium and sodium salts on acidic and alkaline transitions and to thermal transition of cyt c implies a role of hydrophobic interaction between imidazolium cation and polypeptide chain.

Molecular docking of metal ion immobilized ligands to proteins in affinity chromatography.

Immobilized metal ion affinity chromatography (IMAC) has become a widespread analytical and preparative separation method for therapeutic proteins, peptides nucleic acids, hormones, and enzymes. N-Methacryloyl-l-histidine Methyl Ester (MAH) monomer is recently used as a synthesized affinity ligand in IMAC. It is capable of chelating with many transition metal ions such as Zn2+ , Ni2+ , and Cu2+ ions through its histidine residue. In this way, proteins can bind selectively to these immobilized metal ions through MAH as a ligand in affinity chromatography. In this study, we applied the computational docking method on the interactions that occur between the MAH monomer and its complexes with Zn2+ ions as ligands and protein molecules as targets. MAH monomer was drawn and created using the Avogadro software as an optimization tool. Human insulin (Ins) molecule and horse heart cytochrome C (Cyt C) were selected as target proteins to interact with MAH monomer as affinity ligand. Automated docking software AutoDock v4.2 was used for docking of MAH monomer to Ins and Cyt C, respectively. The affinity ligand complexes with Zn2+ ions bound to one, two, and three moles of MAH were studied and compared separately. The lowest binding energies of Ins and Cyt C proteins in 1:1 mol ratio of MAH-Zn2+ were found as (-4.14) and (-4.92) kcal/mol, respectively.

Cytochrome c oxidase oxygen reduction reaction induced by cytochrome c on nickel-coordination surfaces based on graphene oxide in suspension

BackgroundThe electrochemical and spectroscopic investigation of bacterial electron-transfer proteins stabilized on solid state electrodes has provided an effective approach for functional respiratory enzyme studies. MethodsWe assess the biocompatibility of carboxylated graphene oxide (CGO) functionalized with Nickel nitrilotriacetic groups (CGO-NiNTA) ccordinating His-tagged cytochrome c oxidase (CcO) from Rhodobacter sphaeroides. ResultsKinetic studies employing UV–visible absorption spectroscopy confirmed that the immobilized CcO oxidized horse-heart cytochrome c (Cyt c) albeit at a slower rate than isolated CcO. The oxygen reduction reaction as catalyzed by immobilized CcO could be clearly distinguished from that arising from CGO-NiNTA in the presence of Cyt c and dithiothreitol (DTT) as a sacrificial reducing agent. Our findings indicate that while the protein content is about 3.7‰ by mass with respect to the support, the contribution to the oxygen consumption activity averaged at 56.3%. ConclusionsThe CGO-based support stabilizes the free enzyme which, while capable of Cyt c oxidation, is unable to carry out oxygen consumption in solution on its own under our conditions. The turnover rate for the immobilized CcO was as high as 240 O2 molecules per second per CcO unit. General significanceIn vitro investigations of electron flow on isolated components of bacterial electron-transfer enzymes immobilized on the surface of CGO in suspension are expected to shed new light on microbial bioenergetic functions, that could ultimately contribute toward the improvement of performance in living organisms.

Formation of molten globule state in horse heart cytochrome c under physiological conditions: Importance of soft interactions and spectroscopic approach in crowded milieu

To understand protein folding problem under physiological condition, usually taken as dilute aqueous buffer at pH 7.0 and 25 °C, knowledge of properties of folding intermediates is important, such as molten globule (MG). We observed that polyethylene glycol 400 Da (PEG 400) induces molten globule state conformation in cytochrome c at pH 7.0 and 25 °C. This PEG-induced MG state has: (i) native tertiary structure partially perturbed, (ii) unperturbed native secondary structure, (iii) newly exposed hydrophobic patches, and (iv) has 1.58 times more hydrodynamic volume than that of the native protein. Isothermal titration calorimetry and docking studies showed specific binding between PEG 400 and cytochrome c. The study delineates that PEG-protein interactions are more complex than the excluded-volume. The soft interactions need to be seriously studied in crowding milieu that leads to destabilization of protein and overcome stabilizing exclusion volume effect. This study not only can help in unraveling the mystery of steps involved in the proper folding of proteins to solve the massively complicated problems of protein folding but also provides novel insights towards importance of structural change in proteins inside cell where intermediate states of protein import-export easily via membranes rather than native form of proteins.

Refolding of acid denatured cytochrome c by anionic surface-active ionic liquid: Choice of anion plays key role in refolding of proteins

This study was performed to check the effect of temperature on the refolding of acid-denatured horse heart cytochrome c (h-cyt c) at pH 2.0 under the influence of anionic surface active ionic liquid (SAIL) 1-butyl-3-methylimidazolium octyl sulfate ([C4mim][C8OSO3]), various techniques were performed viz. UV–vis, steady-state fluorescence, time-resolved fluorescence, circular dichroism (CD), FT-IR and dynamic light scattering. We found SAIL, [C4mim][C8OSO3] upon addition to the h-cyt c in unfolded state at pH 2.0, stabilized the molten globule (MG), which acts as an intermediate during the protein folding pathway. The investigation of the refolding of h-cyt cin an unfolded state with [C4mim][C8OSO3] shows a surfactant like behavior and stabilizes the MG-like state with the help of electrostatic and hydrophobic interactions. The creation of an MG-like state reveals the presence of a regular structure formed in the presence of SAIL [C4mim][C8OSO3]. The thermodynamic parameters of the refolding of h-cyt c by [C4mim][C8OSO3] were also quantified at various temperatures (25 °C–40 °C). The m value calculated for refolded states of h-cyt c by [C4mim][C8OSO3] shows a noticeable difference. The lower values of m (at higher temperatures) suggest less efficient refolding. This shows the refolding ability of the [C4mim][C8OSO3] decreases with increasing the temperature. Moreover, the anionic SAILs were efficiently utilized in the protein renaturation studies and could be effectively used to solve problems of misfolding and aggregation of proteins.

Electrochemical properties of the interaction between cytochrome c and a hematite nanowire array electrode

We investigate the interaction of horse heart cytochrome c (cyt c) with hematite nanowire array electrodes by cyclic voltammetry to study the electron transfer between redox active proteins and mineral surfaces. Using this model system, we quantify electron transfer rates between cyt c and hematite under varying electric potential and pH conditions. The results are consistent with two cyt c conformations adsorbed at the hematite surface: the native and a partially unfolded form. The partially unfolded protein maintained redox activity, but at a lower redox potential than the native protein. Adsorption of cyt c allowed direct electron transfer between cyt c and hematite, with an interfacial electron transfer rate, k°ET, of 0.4 s−1 for the native form and 0.55 s−1 for the partially unfolded protein at pH 7.07. At pH 4.66, protein adsorption decreased compared to neutral pH and the fraction of partially unfolded protein increased. Additionally, the diffusion controlled electron transfer rate between hematite and the electron shuttling compound anthraquinone-2,6-disulfonate (AQDS) was determined to be k°ET = 8.0·10−3 cm·s−1 at pH 7.07. Modulation of electron transfer rates as a result of conformational changes by redox active proteins has broad implications for describing chemical transformations at biological-mineral interfaces.

The Study of Electron Transfer at the Interface of Cytochromes and Iron Oxide Minerals

Molecular-scale protein-mineral interactions are central to understanding electron transfer mechanisms and microbial processes in subsurface environments. Although, the electron transfer between c-type cytochrome and hematite has been investigated many factors impacting the electron transfer rate have not been determined. We investigated the interactions of horse heart cytochrome c (cyt c) with hematite nanowire array electrodes by cyclic voltammetry to study the electron transfer between redox active proteins and mineral surfaces. Using this model system, specific interactions of cyt c with hematite as a function of electrical potential and pH were examined. We observed direct electron transfer between cyt c and hematite with an interfacial electron transfer rate of kº ET = 0.6 s-1. show buffer pH has an apparent effect on the conformational and adsorption-induced change on the cyt c. Moreover, in the presence of the electron shuttling compound, anthraquinone-2,6-disulfonate (AQDS), electron transfer rate between cyt c and hematite increased to kº ET = 2 s-1, which is more than three times of the rate determined in absence of AQDS. This study provides into biomolecular factors controlling the complex interactions and electron transfer processes between redox-active proteins and minerals, which has broader implications for describing chemical transformations at biological-mineral interfaces at the molecular scale.

Reductive nitrosylation of ferric microperoxidase-11.

Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c, which is considered as a heme-protein model. Here, the reductive nitrosylation of ferric MP11 (MP11(III)) under anaerobic conditions has been investigated between pH 7.4 and 9.2, at T = 20.0°C. At pH ≤ 7.7, NO binds reversibly to MP11(III) leading to the formation of the MP11(III)-NO complex. However, between pH 8.2 and 9.2, the addition of NO to MP11(III) leads to the formation of ferrous nitrosylated MP11(II) (MP11(II)-NO). In fact, the transient MP11{FeNO}6 species is converted to ferrous deoxygenated MP11 (MP11(II)) by OH-- and H2O-based catalysis, which represents the rate-limiting step of the whole reaction. Then, MP11(II) binds NO very rapidly leading to MP11(II)-NO formation. Over the whole pH range explored, the apparent values of kon, koff, and K (= koff/kon) for MP11(III)(-NO) (de)nitrosylation are essentially pH independent, ranging between 5.8 × 105M-1s-1 and 1.6 × 106M-1s-1, between 1.9s-1 and 3.7s-1, and between 1.4 × 10-6 M and 4.6 × 10-6 M, respectively. Values of the apparent pseudo-first-order rate constant for the MP11{FeNO}6 conversion to MP11(II) (i.e., h) increase linearly with pH; the apparent values [Formula: see text] and [Formula: see text] are 7.2 × 102M-1s-1 and 2.5 × 10-4s-1, respectively. Present data confirm that MP11 is a useful molecular model to highlight the role of the protein matrix on the heme-based reactivity.

Cytochrome c electrochemistry on peptide self-assembled monolayers

A short chain peptide is covalently attached to an evaporated gold electrode to form a biocompatible self-assembled monolayer (SAM) surface. The peptide is designed to optimize the interfacial interaction of horse heart cytochrome c (cytc) through the electrostatic interaction of the carboxylate group on the terminal amino acid glutamate and the amine on lysine in cytc. Cytc exhibits quasi-reversible voltammetry and is stable on the time scale of the experiments. Marcus theory is used to calculate the electron transfer rates, which are ~5 times faster for cytc/peptide/Au compared to cytc/11-mercaptoundecanoic acid/Au SAMs of similar chain length. The formal potentials also shift for the cytc/peptide system ~20 mV more positive than cytc adsorbed to 11-mercaptoundecanoic acid-SAM/Au electrodes.

Dynamics of cytochrome c in surface active ionic liquid: A study of preferential interactions towards denaturation

Surface active ionic liquid (SAIL) 1-butyl-3-methylimidazolium octyl sulfate ([C4mim][C8OSO3]) having octyl tail on anionic moiety have been very less investigated with proteins. Herein, we present a study of the structural change of horse heart cytochrome c (h-cyt c) in aqueous solutions of SAIL ([C4mim][C8OSO3]) by optical spectroscopy and molecular docking methods. Time-resolved fluorescence, UV–vis and circular dichroism (CD) spectra indicated that the addition of up to 25 mM of [C4mim][C8OSO3] induces structural changes of h-cyt c resulting from disruption of tertiary structure and after 25 mM of [C4mim][C8OSO3], h-cyt c denatures completely and loses its tertiary structure. Thermodynamic parameters for denaturation of h-cyt c by [C4mim][C8OSO3] were also obtained having ΔG°D and m-value of the unfolded state. The values were found to be lower when compared to the reported for urea denatured h-cyt c. The midpoint concentration of unfolding of h-cyt c by [C4mim][C8OSO3] was found ~25 mM. The results showed that the presence of alkyl chain on the anionic moiety destabilizes the protein. The docking results confirm the preferential interactions of ions of SAIL to residues of h-cyt c and suggest the dominant role of anion [C8OSO3]− by the electrostatic interaction that drives local destabilization of the h-cyt c.

Dynamics of Ionic Liquid-Assisted Refolding of Denatured Cytochrome c: A Study of Preferential Interactions toward Renaturation.

In vitro refolding of denatured protein and the influence of the alkyl chain on the refolding of a protein were tested using long chain imidazolium chloride salts, 1-methyl-3-octylimidazolium chloride [C8mim][Cl], and 1-decyl-3-methylimidazolium chloride [C10mim][Cl]. The horse heart cytochrome c (h-cyt c) was denatured by urea and guanidinium hydrochloride (GdnHCl), as well as by base-induced denaturation at pH 13, to provide a broad overview of the overall refolding behavior. The variation in the alkyl chain of the ionic liquids (ILs) showed a profound effect on the refolding of denatured h-cyt c. The ligand-induced refolding was correlated to understand the mechanism of the conformational stability of proteins in aqueous solutions of ILs. The results showed that the long chain ILs having the [C8mim]+ and [C10mim]+ cations promote the refolding of alkali-denatured h-cyt c. The IL having the [C10mim]+ cation efficiently refolded the alkali-denatured h-cyt c with the formation of the MG state, whereas the IL having the [C8mim]+ cation, which is known to be compatible for protein stability, shows slight refolding and forms a different transition state. The lifetime results show successful refolding of alkaline-denatured h-cyt c by both of the ILs, however, more refolding was observed in the case of [C10mim][Cl], and this was correlated with the fast and medium lifetimes (τ1 and τ2) obtained, which show an increase accompanied by an increase in secondary structure. The hydrophobic interactions plays an important role in the refolding of chemically and alkali-denatured h-cyt c by long chain imidazolium ILs. The formation of the MG state by [C10mim][Cl] was also confirmed, as some regular structure exists far below the CMC of IL. The overall results suggested that the [C10mim]+ cation bound to the unfolded h-cyt c triggers its refolding by electrostatic and hydrophobic interactions that stabilize the MG state.