Abstract
Cytochrome-c (cyt-c), a multi-functional protein, plays a significant role in the electron transport chain, and thus is indispensable in the energy-production process. Besides being an important component in apoptosis, it detoxifies reactive oxygen species. Two hundred and eighty-five complete amino acid sequences of cyt-c from different species are known. Sequence analysis suggests that the number of amino acid residues in most mitochondrial cyts-c is in the range 104 ± 10, and amino acid residues at only few positions are highly conserved throughout evolution. These highly conserved residues are Cys14, Cys17, His18, Gly29, Pro30, Gly41, Asn52, Trp59, Tyr67, Leu68, Pro71, Pro76, Thr78, Met80, and Phe82. These are also known as “key residues”, which contribute significantly to the structure, function, folding, and stability of cyt-c. The three-dimensional structure of cyt-c from ten eukaryotic species have been determined using X-ray diffraction studies. Structure analysis suggests that the tertiary structure of cyt-c is almost preserved along the evolutionary scale. Furthermore, residues of N/C-terminal helices Gly6, Phe10, Leu94, and Tyr97 interact with each other in a specific manner, forming an evolutionary conserved interface. To understand the role of evolutionary conserved residues on structure, stability, and function, numerous studies have been performed in which these residues were substituted with different amino acids. In these studies, structure deals with the effect of mutation on secondary and tertiary structure measured by spectroscopic techniques; stability deals with the effect of mutation on T m (midpoint of heat denaturation), ∆G D (Gibbs free energy change on denaturation) and folding; and function deals with the effect of mutation on electron transport, apoptosis, cell growth, and protein expression. In this review, we have compiled all these studies at one place. This compilation will be useful to biochemists and biophysicists interested in understanding the importance of conservation of certain residues throughout the evolution in preserving the structure, function, and stability in proteins.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-013-1341-1) contains supplementary material, which is available to authorized users.
Keywords: Cytochrome-c, Key residues, Folding and stability, Natural selection, Electron transport chain, Apoptosis
Introduction
Cytochrome-c (cyt-c) is a small (12.4-kDa) globular protein. It is vital for living organisms due to its potential role as penultimate electron transporter in aerobic as well as anaerobic respiration (Fig. 1) [1, 2]. Cyt-c, otherwise present in the intermembrane space when released in cytosol, plays an indispensable role in apoptosis [1, 3, 4] (Fig. 1). It has also been shown to have antioxidant [5–7] and peroxidase activity [8–14]. Electron transport property of cyt-c is well established in both prokaryotes and eukaryotes. However, apoptosis has been reported to be essentially required for the survival of eukaryotes. It seems that these two properties of cyt-c have evolved separately along the evolutionary scale [15]. Besides its significant role in electron transport, cyt-c is considered as a model protein for studying folding and stability [16–27] because of ease in its preparation, extraction, and its capacity to accept genetic alteration in its genome [28–32]. Oxidation state of heme and conformational changes in heme crevice are supposed to trigger pro-apoptotic activity of mitochondrial cyt-c [9, 33–36].
Fig. 1.
Cyt-c participating in electron transport chain (ETC) and apoptosis. ETC I–IV refers to complexes involved in ETC. Complexes I (NADH dehydrogenase) and II (succinate dehydrogenase) use electrons to reduce coenzyme Q, which transfers these electrons to complex III (cytochrome bc 1 complex). Cyt-c receives electrons from complex III and shuttle them to complex IV (cyt-c oxidase), which in turn use them to reduce molecular oxygen to water. Apoptosis: On receiving an apoptotic stimulus, cyt-c gets released from mitochondria into the cytosol. In cytosol, cyt-c takes part in apoptosome formation and procaspase-9 activation, which further activate effector caspases, eventually causing cell death
Owing to their robustness, stability, as well as their capability to reversible denaturation, cyts-c represent an ideal system for protein folding studies [37–47]. The protein folding mechanism has always been a perplexity in structural biology, which gave rise to Levinthal’s paradox in late 1960s and it is still not completely understood [48–57]. Structural heterogeneities in the native, unfolded, and intermediate states of a protein suggest the existence of multiple folding pathways [54, 58–62]. Cyt-c has been shown to have varied opinions regarding its folding mechanism. Travaglini-Allocatelli et al. [63] proposed that in spite of large differences in their physico-chemical properties and thermodynamic stability, cyts-c share a consensus folding mechanism. On the other hand, various reports that confirm different folding pathways for closely related species of cyt-c are also available [64, 65]. Equilibrium studies on horse cyt-c (h-cyt-c) suggest a rather complex stepwise folding process [37, 38, 42, 66–75]. While bovine cyt-c (which differs from h-cyt-c at only three residue positions) have been shown to follow different folding pathway [64, 65, 76]. Furthermore, yeast cyt-c (y-cyt-c) displays a high degree of heterogeneity in the denatured state with both extended and compact structures present together [77–82]. Various cytochromes have been shown to involve formation of partially structured intermediates along the course of folding [17, 83–87]. Molten globule is one of the most characterized intermediates in case of cyt-c along with other reported intermediates [47, 65, 67, 88–92].
Factors governing the stability of the protein are hydrogen bonding, electrostatic interaction, hydrophobic interaction, van der Waals interaction, disulfide bonds, and relative conformational entropy [93]. Stability of cyt-c could be studied as local stability, which could be measured by determining the Met80–Fe(III)–heme interaction (695-nm absorbance band/416 CD band) [94, 95] or alkaline pKa [96, 97] and global stability, measured by denaturation studies [98]. The redox potential, which decides the ability of cyt-c as an electron transporter, is determined by axial ligation [99], measuring the polarity of heme [100], heme solvent accessibility [101], heme propionate solvation [102], and electrostatic interaction [103–105]. The oxidation state of the protein is also important in determining the functional properties of cyt-c. The sequence and stability analysis revealed that the contribution of individual amino acids to the stability and functional properties of a specific protein is sometimes difficult to assess, since it may not be possible to design substitutions that eliminate one type of interaction without simultaneously affecting other types of interactions, and in turn, function [93]. Hence, the analysis of combined effect of mutations will be more precise [106].
Though the tertiary structure of cyt-c has almost been conserved throughout the evolution (Fig. 2), the stability of the cyt-c from different species varies. Cyt-c retains its function and structure even with more than 40 differences in the sequences of the horse and yeast cyts-c. However, only three differences in the primary structure of the bovine and horse cyts-c lead to considerable differences in the folding mechanism and stabilities of the two proteins [22, 27, 64, 107]. Interestingly, alterations that significantly affect stability of cyts-c are due to those residues that are invariant or highly conserved [108]. Apart from heme-binding, residues such as Cys14, Cys17, His18, and Met80 (numbering of the residues is done with reference to the vertebrate numbering system), there are many positions in cyts-c, such as Trp59, Tyr67, Phe82, Pro30/71/76 Asn52, etc., which are conserved in almost all subfamilies. Crystal structure analysis has shown that certain residues such as Gly6, Phe10, Leu94, and Tyr97 are also conserved and form many significantly strong contacts (23 interatomic contacts) between N- and C-terminal helices contributing to the stability of the folded protein and hence are quite important for the folding of protein [109–113].
Fig. 2.
Overall structure of horse cyt-c showing conserved residues in ball-and-stick model. Structure was drawn using the atomic coordinates of PDB id 1HRC [111]
Comparison of the predicted folding nucleus with experiments can be performed by the combination of site-directed mutagenesis with kinetic studies [114]. Recently, we have observed a drastic change in the structure and stability of h-cyt-c on mutation of Lue94 to Gly94 [88]. Although other mutations (Leu94Ile and Leu94Val) do not alter the stability of the native state of h-cyt-c, a twofold decrease in the folding rate and a large decrease in the population of molten globule-like intermediate have been observed in these mutants [115]. These findings further suggest that the conserved residue Leu94 is essential for the proper folding of cyt-c [88, 116]. Marmorino et al. [117] showed that Phe10Tyr, Phe10Trp, Leu94Ile, and Tyr97Phe mutants of yeast iso-1-cytochrome c destabilize its acid state exactly to the same extent as they destabilize the native state.
One of the most interesting features of cyt-c is that it retains its structure and function even after enormous variations in its polypeptide sequence [89]. Hence, it might be possible that the purpose of protein moiety is mainly to wrap the heme (a constant part), which enables selectivity in partner recognition as well as tune the reduction potential of the iron ion. Therefore, the large variations observed in the evolution of cyt-c is to optimize the interaction and electron transfer. Comparative studies of various cyts-c, combined with investigations of the effects of mutations on protein stability, have provided some general suggestions on how to enhance the stability of proteins [64, 118–120]. Few residues are highly conserved throughout the evolution and these residues are termed as “key residues”. In this review, our aim is to focus on the role of key residues in cyt-c from eukaryotes in protein folding, stability, and function.
Out of all sequences reported in UniProt (www.uniprot.org), 285 sequences that are either manually reviewed or have their genes termed as “cyc” have been considered (see Supplementary figures and tables). Multiple sequence alignment suggests high levels of sequence similarity among all sequences ranging from 28 to 99 % as compared to h-cyt-c (Supplementary Table S2). Surprisingly, out of 104 ± 10 amino acid residues (as reported in most of the cyt-c sequences), a few residues are highly conserved in all these proteins, revealing their role in structure, function, and stability (Table 1). Out of 104 amino acid residues, only one residue, Cys17, is identical in all sequences of cyt-c. Furthermore, few residues are also highly conserved in all cyts-c, including Gly6, Phe10, Cys14, His18, Pro30, Trp59, Tyr67, Pro71, Lys72, Pro76, Thr78, Met80, Leu94, and Tyr97. Structure analysis further suggests the precise role of these residues in maintaining the structure and stability of cyt-c (Table 1; Fig. 2). In this review, we have categorized conserved residues into various groups and present an extensive analysis for their role in folding, stability, and functions of cyt-c.
Table 1.
List of conserved residues important for function and stability of cyts-c
| S. no. | Positionsa | Residue | Alternate residues |
|---|---|---|---|
| 1 | 6 | Gly | Asp, Ala |
| 2 | 10 | Phe | Tyr, Ser, Ala |
| 3 | 14 | Cys | Ala |
| 4 | 17 | Cys | – |
| 5 | 18 | His | Arg |
| 6 | 29 | Gly | Ala |
| 7 | 30 | Pro | Gln, Xb |
| 8 | 32 | Leu | Gln |
| 9 | 41 | Gly | Val, Arg, Ala |
| 10 | 48 | Tyr | Phe, His |
| 11 | 59 | Trp | Tyr |
| 12 | 67 | Tyr | Phe, Trp |
| 13 | 68 | Leu | Ile, Trp |
| 14 | 71 | Pro | Ala |
| 15 | 72 | Lys | Arg, Met, Gln, Ser, X |
| 16 | 76 | Pro | Lys |
| 17 | 77 | Gly | Lys, Asn |
| 18 | 78 | Thr | Asn, Gly |
| 19 | 79 | Lys | Asn |
| 20 | 80 | Met | Thr |
| 21 | 82 | Phe | Leu, Tyr |
| 22 | 94 | Leu | Ile, Val |
| 23 | 97 | Tyr | Phe, Trp, Ser |
aNumbering based on the sequence of h-cyt-c
bX is any amino acid
Residues forming covalent linkages to the heme
Cys14/17
Structure and biochemical studies suggest that cyt-c has two cysteine residues, Cys14 and Cys17, which form thioether bonds to the second and fourth vinyl side chains of the heme, respectively [111, 112]. These cysteine residues are a part of a pentapeptide motif –Cys–Xxx–Xxx–Cys–His– (CXXCH), which is highly conserved in most of the organisms with a few exceptions where the motif is a bit altered [121–124]. Covalent attachment of heme to polypeptide is the characteristic feature of c-type cyts, rendering it to be different from other cyts (e.g., cyt-b) [1, 2]. The covalent attachment of heme to CXXCH motif of apo-cyt-c is the critical step of cyt-c maturation [125, 126]. This pentapeptide motif, along with covalently bound heme, is suggested to serve as a basic entity for the rest of the polypeptide chain to fold around [127], so any change in this entity is expected to alter the structure and the folding pattern of the protein. Furthermore, covalent attachment of heme has many functional and structural implications. It enhances axial ligand strength of heme, contributes in electronic environment of heme crevice, set up redox potential, and add robustness to the structure of cyt-c preventing the loss of heme during large conformational changes [125, 128]. Mutation of Cys14/17 with Tyr/Phe/Trp prevented the growth of Saccharomyces cerevisiae (D311-3A strain carrying cyt-c gene mutated for Cys14/17) on lactate medium [28, 129], suggesting absolute involvement of these residues in proper folding of the protein polypeptide chain, which is required for the desired function of cyt-c [130].
Sequence alignment of cyts-c showed that Cys17 is completely conserved throughout the evolution, whereas Cys14 is found to be highly conserved with a few exceptions (Figure S1). Hampsey et al. [28] suggested the role of these cysteines in translocation of cyt-c. It is known that the apoform is initially synthesized in the cytoplasm and is subsequently translocated to the mitochondrion where heme gets covalently attached by the cyt-c synthetase to yield mature holo-cyt-c [131]. It was further suggested that some amino acid replacements might prevent translocation, thereby precluding folding of the protein to its mature form. In general, the translocation of cyt-c is presumably prevented by replacements of the Cys14 or Cys17 residues [28]. Failure of heme incorporation in protein variants lacking Cys14/17 and their simultaneous accumulation in the inter-membrane space of mitochondria also suggest their significant contribution in holo-cyt-c formation [132–134]. Mavridou et al. [134] suggested that for the cyt-c maturation system for heme binding, CXXCH motif is the authentic substrate, and stereochemically favored in vivo. Mutants with only single Cys (CXXXH or XXXCH) are very poor substrates. Rosell and Mauk [130] suggested the role of these cysteines in expression of cyt-c, as CXXCH motif in cyt-c, is an essential component for the expression of mitochondrial cyts-c. A decreased expression may be a consequence of poor recognition of the protein molecule as a substrate for the cyt-c heme lyase, and therefore it reduces the efficiency of catalysis of heme attachment and stability of the variant in vivo. Cys17, an absolute invariant residue, is essentially required for the covalent heme attachment. Expression of Cys17Ser mutant showed an insignificant yield (<0.1 mg/l) as compared with the normal yield (20 mg/l). This yield is also lower than that achieved with the Cys14Ser variant (2 mg/l) [130]. No modifications in culture conditions could improve the yield of isolated protein. Absence of any naturally occurring variant for this residue also signifies its importance.
Cys14 is highly conserved except in mitochondrial cyt-c of some protozoan species where Cys at the N-terminal of –CXXCH– (i.e., Cys14 analogue) is replaced by Ala [125]. A Cys14Ser mutant of y-cyt-c was prepared, and it was observed that the 695-nm absorption band, a characteristic of Met80–heme interaction in the wild-type (WT) protein, was found missing even in the mild acidic condition [130]. One of the conclusions of this study is that the elimination of this thioether bond to heme resulted in the electronic perturbation that shifts this transition to higher energy where it is more difficult to detect. It was further concluded that for y-cyt-c, both the coordination of Met80 residue to Fe(III) and the presence of the thioether bond between Cys14 and the heme second-vinyl are required for observation of the weak charge-transfer band near 695 nm. Cowley et al. [135] showed that transligation of Met80 is energetically more favorable in cases where both thioether bonds are formed rather in the presence of a single covalent bond. It has been shown that the formation of Met80–Fe bond in cyt-c requires a certain level of restructuring, which is offered by two covalent bonds formed between cysteines (Cys14 and Cys17) and the heme [135, 136]. Conformational constraints due to these heme-peptide linkages energetically favor His-Fe coordination and folding of cyt-c. These covalent linkages also strengthen the intrinsic ligand field of His18 and also assure a low spin configuration for ferric porphyrin [135]. Cys14 and/or Cys17 also contribute(s) to the stability of the native molecule (2 kcal/mol) by virtue of conformational restriction provided by the thioether bonds leading to reduction in the entropy of the molecule [137].
Axial ligands of heme
Numerous structural and biochemical studies have shown that covalently attached heme along with its axial ligands are essentially important for structure, stability, and function of cyt-c, and play a significant role in the folding process [94, 138–144]. Covalently attached heme also requires its axial ligands to let the polypeptide attain its functional structure [145–147]. His18 and Met80 are the two axial ligands of cyt-c, where His18 binds the heme from the proximal front (right side of the molecule as mentioned by Takano) and Met80 is present on the distal side (left side) [148, 149]. Among these axial ligands, the Met80–Fe(III) bond is labile and dissociates more rapidly, even under mild conditions [96, 150–154], while His18 remain bound to heme even in typical denaturing conditions [155–159].
His18
His18 is a highly conserved residue, acts as a fifth ligand to the heme, and forms an axial coordination bond with the heme iron [28, 129, 160]. This coordinate bond (between its ε-nitrogen and the heme iron) is projected in such a way that the imidazole ring lies almost perpendicular (76°) to the plane of the pyrrole nitrogen atom of the heme, and it skips any steric contact with the heme moiety [111]. Allen et al. [161, 162] suggested a critical role of His, the proximal ligand of heme, in kinetics of heme attachment, supposedly by facilitating the thioether bond formation between cysteine and heme. Moreover, His18 directly stabilizes the heme within the protein structure and is involved in the electron transfer mechanism [161, 162]. The anionic character of His18 plays an important role in determining the stability of different oxidation states, redox potential, and reactivity of cyt-c [128, 163–165].
Any mutation at this position would be expected to abolish the function of cyt-c. Various studies on cyt-c suggest that His18 remains bound to the oxidized heme iron under typical denaturing conditions, such as in concentrated guanidinium chloride (GdmCl) or urea solution near pH 7.0 [155–158]. The conformational stability of this bond could be attributed to many reasons. Since it is a part of pentapeptide motif CXXCH, it strengthens the heme–protein interaction along with covalently linked cysteine. Presence of an H-bond between backbone NH of His18 and the carbonyl of Ala15, which is another residue of the pentapeptide motif in h-cyt-c, also supports the stability of this axial ligand [111]. The presence of a persistent single highly protected backbone amide of His18 as observed in H-exchange experiment, suggests the retained ligation of His18 to heme in the unfolded state of cyt-c [166]. All these findings clearly explain the significant contribution of His18 in the folding and stability of cyt-c and the reason as to why His18 remains bound to heme even in denaturing conditions.
Site-directed mutagenesis studies in the yeast showed the inability of His18Ala mutant to grow because of the formation of non-functional cyt-c [166]. The absence of peak at 548 nm (α peak), in the difference spectrum (between reduced and oxidized cells) measured in low temperature difference spectroscopy of intact cells, an indicator of holo-protein formation, denote the role of His18 in the function of cyt-c [166]. Revertant colonies, obtained by random mutagenesis of His18Ala mutants with synthetic oligos (that randomizes the codon for His18), were found to have only His at position 18, even when all the other 19 oligos had equal chances to integrate at this position and produce viable colonies. This observation clearly indicates that only His can act as a ligand at this position [166].
Met80
Met80 is also a highly conserved intrinsic ligand of heme and binds axially to the iron atom [111, 112]. The coordination bond between heme and its sixth ligand, though labile, plays a crucial role in maintaining the oxidation–reduction potential, electron transport, structure, and conformational stability of cyt-c [139, 145, 167, 168]. Under the physiological conditions, Met80 is found responsible for the relatively high reduction potential of cyt-c and favors the protein’s reduced state [99, 139, 169–171]. The disruption of this heme Fe(III)-Met80 axial bond leads to a drastic lowering of cyt’s redox potential [139, 170, 172] as the protein rearranges within [173, 174]. Spectroscopic diagnostic tool for this Fe-Met80 coordination bond, 695-nm absorption band, also provides valuable information about heme pocket region and redox properties of cyt-c [127].
Mutant Met80Cys in h-cyt-c [139] and Met58Cys (Met58, sixth axial ligand in Porphyra yoezensis cyt-c 6, is an analogue to Met80 of h-cyt-c) [145] showed a remarkable decrease (~600 mV) in their reduction potential as compared to their (WT) forms. Mutants such as Met80Leu [139], Met58His [145], and Met80His [170] also showed a drop in their reduction potentials. Kinetics studies has also revealed an important role of Met80 as sixth heme axial ligand in folding-unfolding of the protein [166].
Many mutants of Met80 have been studied for their functional relevance. It has been observed that in most of the cases, the replaced residue (chemically modified or mutated) at position 80 is unable to form axial coordination with heme rendering the mutant penta-coordinate, e.g., Met80Ala mutant in both yeast- [138] and h-cyt-c [153, 175]. Such penta-coordinate mutants either stay vacant or bind small exogenous ligands such as dioxygen or carbon monoxide (CO) [138, 141, 142, 176, 177]. Ferrous-CO adduct has shown to have blocked the electron transfer as CO strongly stabilizes the complex formed between ferrous-CO adduct and its redox partner [142]. This explains the contribution of Met80 in providing the right environment/channel for electron transfer.
Mutation at position 80 also leads to structural distortion, which is pK-dependent and varies with the type of residue replaced at position 80 [142]. Substitution with a hydrophilic residue alters the apparent pK value, adds occupied volume as well as charge permitting disruption in structure allowing easier rearrangement of the loop [142]. On the contrary, Ala (Ser) in Met80Ala (Met80Ser) mutant, being a small uncharged molecule allow heme pocket to retain its hydrophobicity preserving the structure [141, 142] similar to that of the native molecule [112, 178]. This structural distortion also affects the apparent quantum yield for CO photo-dissociation in ferrous-CO adducts of Met80 mutants. Met80Ala-CO adduct due to its tight structure around the heme, does not allow CO to dissociate on photolysis, thus preventing the electron transfer to cyt-c oxidase along with high yield of geminate recombination [141, 142]. While in case of Met80Asp and Met80Glu the easier rearrangement around the cavity allows the quantum yield of photo dissociated carbon monoxide to be ten times higher than that of Met80Ala [142]. Structural changes in the mutants do not influence the recognition of the cyt-c for its redox partner. The interaction between cyt-c and its oxidase, remains very tight, but low quantum yields make them less than ideal for use as light-induced electron donors [142]. Casalini et al. [179] have also shown the effect of replacement of Met80 with Ala on the thermodynamics and kinetics of ferric to ferrous reduction in cyt-c.
Satoh et al. [145] have also shown the role of heme axial ligand in conformational stability of cyt-c. Denaturation studies in cyt-c 6 of Porphyra yezoensis [145] and h-cyt-c [44, 167] have shown that the dissociation of the sixth ligand (Met80) is tightly coupled with unfolding of the protein. An increase in ∆G D (Gibbs free energy change on denaturation in the absence of denaturant) for mutant Met58Cys (Table 2) in cyt-c 6 is observed, which Satoh et al. [145] attributed to the formation of a stable bond between Fe and Cys with secondary structure (74.6 %) similar to that of the WT (73.8 %). Theoretical ab initio energy calculation has also shown that His-Fe and Cys-Fe bonds are more stable than Met–Fe bond [145]. Similarly, an increased ligand field strength was observed in Met80Cys of h-cyt-c [140]. Contrary to Met58Cys, Met58His showed a decrease in thermodynamic stability, which is accredited to the reduction in the secondary structure (66.1 %). This destabilization is believed to be due to the exposure of hydrophobic residues to solvent [145]. Indiani et al. [180] have shown the effect of the sixth axial ligand on heme conformational changes in two different cytochromes one of which has His as its sixth ligand (Methylophilus methylotrophus cyt-c) unlike the other, h-cyt-c.
Table 2.
Effect of mutation on the Gibbs free energy of stabilization of cyt-c
| Positiona | Organism | Protein | Mutation | ΔΔG bD (kcal/mol) | Reference |
|---|---|---|---|---|---|
| Phe10 | Yeast | iso-1-cyt-c | Ile | −1.14 | [187] |
| Met | −3.52 | [187] | |||
| Trp | −1.30 | [187] | |||
| Tyr | −0.47 | [187] | |||
| CcSMe | −3.35 | [182] | |||
| Trp59 | Rhodobacter capsulatus d | Cyt-c 2 | Tyr | −2.50 | [228] |
| Tyr67 | Rat | cyt-c | Phe | −1.96 | [98] |
| Yeast | iso-1-cyt-c | Phe | +1.30 | [238] | |
| Yeast | iso-1-cyt-c | Phe | +1.5 | [237] | |
| Pro71 | Yeast | iso-1-cyt-c | Val | −1.00 | [281] |
| Thr | −1.70 | [281] | |||
| Ile | −1.70 | [281] | |||
| Yeast | iso-2-cyt-c | Thr | −0.80 | [269] | |
| Pro76 | Yeast | iso-1-cyt-c | Leu | −3.44 | [287] |
| Gly | −0.78 | [287] | |||
| Val | −1.07 | [287] | |||
| Arg | −0.97 | [287] | |||
| Ser | −1.89 | [287] | |||
| Tyr | −2.07 | [287] | |||
| Trp | −2.38 | [287] | |||
| Yeast | iso-2-cyt-c | Gly | −1.19 | [282] | |
| Met80 | Porphyra yezoensise | cyt-c 6 | Cys | 3.02 | [145] |
| His | 0.95 | [145] | |||
| Phe82 | Yeast | iso-1-cyt-c | Trp | −0.50 | [252] |
| Tyr | −0.20 | [251] | |||
| Leu | −0.20 | [251] | |||
| Ile | −0.40 | [251] | |||
| Ala | −0.70 | [251] | |||
| Ser | −0.80 | [251] | |||
| Gly | −1.00 | [251] | |||
| Leu94 | Yeast | iso-1-cyt-c | Ile | 0.33 | [187] |
| Val | −1.06 | [187] | |||
| Thr | −3.05 | [187] | |||
| Ala | −3.58 | [187] | |||
| Horse | cyt-c | Gly | −4.84 | [88] | |
| Ile | 0.20 | [115] | |||
| Val | −0.80 | [115] | |||
| Ala | −3.50 | [115] | |||
| Tyr97 | Yeast | iso-1-cyt-c | Ala | −4.87 | [187] |
| Phe | 0.24 | [187] |
aNumbering based on the sequence of h-cyt-c
bΔΔG D is the difference between ΔG D(mutant) and ΔG D (wild type) where ΔG D is the value of Gibbs free energy measured under the native condition
cS-methylcysteine
dResidue analogous to Trp59 of h-cyt-c is Trp67 in Rhodobacter capsulatus
eResidue analogous to Met80 of h-cyt-c is Met58 in Porphyra yezoensis
Thus, we can conclude that axial Met in cyt-c is important in maintaining the heme reduction potential, thermodynamic stability, structure, and electron transfer property of the protein. Met80-external ligand adducts could be useful in studies of long-range donor–acceptor electronic couplings in proteins.
Conserved residues in N- and C-terminal helices
Examination of crystallographic [112, 181] and NMR [182, 183] data of cyts-c shows that the N- and C-terminal helices interact in a specific manner (Fig. 3). Perpendicular coupling of these helices is a remarkable feature of most conserved structural motifs in both prokaryotic and eukaryotic cyts-c [181]. The helix–helix interaction is implicated in early kinetic folding intermediates [115, 184]. Other proteins that exhibit such specific association of helices include DNA binding proteins [185, 186]. The helix–helix interface is a structurally and functionally essential motif providing significant information about equilibrium thermodynamics of thermal denaturation [115, 184, 187]. Likewise, the N- and C-terminal helices of cyt-c have been a model system for the study of this structural motif [184, 188].
Fig. 3.
N- and C-terminal interface showing close interaction of conserved residues
Unlike other important motifs, the interface neither takes part in electron transport nor is a part of interacting domains involved in the complex formation between cyt-c and its partners [189]. Instead, association of N- and C-terminal helices represents an early event along the folding pathway [166, 184, 190], suggesting that the interaction of the helices directs folding. The helices interact with each other as soon as the heme is covalently bound to the polypeptide via thioether bonds at the end of the N-terminal helix [191]. It is assumed that they are involved in a common folding nucleus of all subfamilies of c-type cyts. Hence, alteration of the interface may directly affect the folding, heme attachment, and mitochondrial import, thus indirectly affecting the function of cyt-c [23].
The interface formed by the packing of the N- and C-terminal helices involves the conserved residues Leu94 and Tyr97 from the C-terminal helix in specific interaction with Gly6 and Phel0 from the N-terminal helix, respectively [192]. The side chain of Leu94 packs into the hole created by Gly6 in a peg-in-hole interaction while Tyr97 and Phe10 pack against each other through an aromatic–aromatic interaction. On the basis of the sequence alignment, among these four interacting residues, Gly6 is the most conserved residue with only one natural variant that has Asp at position 6, while Phe10 has many variations. Leu94 is also well conserved, with two different amino acids (Val and Ile) as its natural replacement while Tyr97 is also present in most of the cytochromes. These sets of conserved non-functional residues at the interface between N- and C-terminal helices suggest that these four residues may be of special importance for the structure and/or the folding of cyt-c. To generate variants, amino acid residues (Gly6, Phe10, Leu94, Tyr97) at the interface of N- and C-helices were mutated [23, 115, 188, 193]. The structure, function, and stabilities of many variants have been determined [23, 115, 187, 194].
N-terminal helix
Gly6 is present at the region of closest approach between the N- and C-terminal helices. Auld and Pielak [188] used in vitro random mutagenesis to produce 16 functional missense mutants at Gly6 and/or Phel0 of the interface, including the two natural substitutions Gly6Asp and Phel0Tyr [195, 196]. Previous belief that this interface is very tightly packed [149], got under speculation when various mutants (Gly6Ser, Gly6Asp, Gly6Val, Gly6Met) at this position gave rise to a functional cyt-c signifying flexibility in the packing around this residue [188]. Auld and Pielak [188] in their statistical analysis showed a probability of 0.74 for all functionally attuned amino acids to be present at this position. However, all the functional mutants produced, except Gly6Ala grew at a rate indistinguishable from WT and showed much more reduced and temperature-sensitive growth [28, 188].
Invariance of Gly6 cannot be accredited to α-helical stabilization, because amino acid substitutes at this position are not disrupting the helix. Thus, helix stabilization cannot be the reason for its conservation at this position. In fact, it has been shown that amino acids, other than Gly and Pro are more stabilizing towards helix formation [197–200]. The most probable reason that Gly6 is conserved is the lack of space to accommodate a side chain of any other amino acid at this position, where Leu94 interacts with Gly6 [149]. Furthermore, Gly6 creates a room for side chain of Leu94 to satisfy constraints producing stable helical packing. Temperature sensitivity and the reduced growth of the variants might be due to unfavorable interactions of their side chains with the C-terminal α–helix [188].
In case of Phe10, the flexibility of producing mutants with replacement at position 10 with growth similar to that of the WT was observed to be more. Substitution with Tyr, Trp, Leu, Ile, Val, Met, and Cys at position 10 showed normal growth [188]. However, the thermodynamic study conducted by Pielak et al. [187] showed a decrease in thermodynamic stability of the mutants (Table 2). A considerable characteristic which all functional Phe10 mutants displayed was preservation of hydrophobicity at position 10 [188]. Like Gly6, around 75 % of the substitutions have produced partially functional mutants, but unlike Gly6 functional variants, broad range of side chain volume can be accommodated at this position (ranging from an increase of 38 Å3 for Trp to a decrease of 80 Å3 for Cys). Substitutes that further decrease the volume (Phe10Ser, Phe10Gly) or basic residues (Phe10Arg) resulted in abolition of function [188].
Conformational shifts in N- and C-helices allow flexibility at interhelical interfaces. Auld and Pielak [188] suggested that few substitutes, which produced functional mutants (e.g., Gly6Ala, Phe10Tyr, and Phe10Leu), and are also present in natural variants of cyt-c, are probably accommodated by the reorganization of side-chain and small changes in the helix–helix torsion angle. However, larger shuffling (e.g., Phe10Cys and Gly6Met) may alter the heme pocket [201]. Therefore, residues at position 6 and 10 are required to maintain the stability and proper orientation of the helices, hydrophobicity of the core, and for allowing the proper folding of the protein [184]. In addition, two mutants (Phe10Cys and Phe10Met) possess sulfur-containing amino acids. Aromatic–aromatic and aromatic–sulfur interactions have been shown to stabilize proteins considerably [202, 203]. Recently, Phe10 has also been suggested to be a recognition site for the cyt-c heme lyase, an enzyme involved in cyt-c maturation of higher organisms [204].
C-terminal helix
The C-terminal helix is more lenient for substitutions than is the N-terminal helix [23]. A total of 30 % of all the 400 possible substitutions at positions 94 and 97 produced functional proteins [23]. There is a greater allowed range of motion for the C-terminal helix and shows high degree of shift with respect to the heme because it is more stable in nature [201]. Many variants of Tyr97 have been studied [23, 187]. Mutants Tyr97Phe, Tyr97Glu, Tyr97Cys, Tyr97Asn, and Tyr97Ala showed growth on lactate medium similar to that of the WT [23]. Thermodynamic study of Tyr97 variants has shown that Tyr97Phe is one of the few variants (out of these four residues of terminal helices) which show an increase in stability (Table 2) [187]. This observation is supported by the fact that Phe is better suited to the protein interior and favorably pairs with other Phe, thus providing a stabilizing effect [202]. Moreover, substitution of Tyr97 with Arg or Lys resulted in loss of function. In terms of polarity and charge, only negatively charged residues were able to produce functional variants. While positively charged residues (Tyr97Arg, Tyr97Lys) rendered mutant protein functionless [23]. Similar results have been reported for substitutions at Gly6 and Phe10 [188].
Tyr97 has been suggested to have a role in apoptosis in mammalian cyt-c [205, 206]. Tyr97 lies on the surface region of cyt-c, which acts as a site for the binding to hydrophobic moieties (e.g., cardiolipin in inner mitochondrial membrane). On receiving the apoptotic stimulus, cyt-c gets released from the mitochondria into cytoplasm. Tyr97 takes part in oxidation of cardiolipin, thus allowing detachment and release of cyt-c from mitochondria to cytoplasm to trigger apoptosis [205].
Like Phe10, functional mutants at positions 97 exhibited broad volume range varying from −0.2 Å3 to −112 Å3 (Tyr97Phe to Tyr97Ala) [23]. All the functional missense mutants can somehow be linked to weak polar interaction with Phe10. Examination of the double missense mutant (at position 94 and 97) showed that one hydrophobic residue is required at either position to maintain function [23].
Leu94 is a highly conserved residue interacting with Gly6, Ile9, Phe10, Leu68, Ile85, Gln90, Arg91, and heme via van der Waals interactions that occur between N- and C-terminal helices [88, 111]. Like Phe10, Leu94 is solvent-inaccessible and present in the peg-in-hole interaction with Gly6. Fredericks et al. [23] carried out random mutagenesis and observed ten variants with substitution at position 94 to be functionally compatible. Mutants with substitution of Phe/Val/Ser/Thr/Cys/Met/Ile at position 94 had a growth rate similar to that of WT while the rest were either temperature sensitive or did not grow. Ile and Val are also present at this position naturally in some organism (Figure S1). The volume range obtained for the functional variants at position 94 varies between +36 Å3 to −101 Å3 (Leu94Phe to Leu94Gly). Neither positively charged nor negatively charged residue (unlike Tyr97) replacements were found to be functional at this location. Even helix propensity alone cannot be considered responsible for the associated stability changes. If this would have been the case, Ala and not Ile at position 94 should have stabilized the helix [199], as observed in position 94 variants (Table 2). Discrepancies in ∆G D values between double mutants (Leu94Ile;Tyr97Phe or Leu94Ala;Tyr97Phe) and sum of individual ∆G D of corresponding single mutants (e.g., Leu94Ile and Tyr97Phe) suggested that it is a cooperative structural as well as thermodynamic interaction between helices that stabilizes the protein [106, 207].
Variants with smaller residues (Ala/Gly) substituted at position 94, though produced functional protein, were reportedly quiet less stable (Table 2). Leu94Gly showed a dramatic drop in the thermodynamic stability; ΔG D of the mutant decreased by about 5 kcal mol−1 [23, 88] (Table 2). Single substitutions when induced together to produce a double mutant may have either compensating effect (i.e., one functional substitution may compensate for the loss of function caused by the other) or may have an additive or synergistic effect. For instance, in case of Leu94Ala; Tyr97Phe, the apparent destabilizing effect of mutation at position 94 (Leu94Ala is temperature sensitive), was overcome by simultaneous substitution of Tyr97 with Phe retaining the properties similar to that of WT. On the contrary, single-functional (e.g., Leu94Phe, Tyr97Asn) or partially functional (e.g., Leu94Asn, Tyr97Thr) variants when brought together rendered the double mutant (e.g., Leu94Phe; Tyr97Asn) non-functional [23]. This type of functional effect has also been noted in second site revertants [208], including those found in different areas of y-cyt-c [209, 210].
Equilibrium and kinetic studies of Leu94 mutants revealed its role in the formation of early and late intermediates (such as molten and pre-molten globule) that are formed during folding of protein [71]. A-state (state of the protein at low pH) [150, 158, 211–217] attained for the mutant Leu94Val, is quiet similar to that formed for the WT [71]. A-state represents an equilibrium analogue of the late folding intermediate [71]. In contrast, Leu94Ala, which was shown to have properties similar to that of pre-molten globule [218, 219], assumes an A-state closely related to early folding intermediates [71]. Recently, we observed that Leu94Gly mutant has all the common structural characteristics of a molten globule [88]. A premolten globule state was also observed during unfolding of Leu94Gly [67]. Marmorino et al. [83, 117] have also shown that interaction of N- and C-terminal helices is crucial in stabilization of the A-state. Thus, it could be assumed that Leu94 is there to play an important role in folding of cyt-c. In summary, the N- and C-terminal helices interact both structurally and thermodynamically. These interactions are important events in the course of folding of the protein to its native state.
Aromatic amino acids
Trp59
Trp59 is another highly conserved residue and, in most of the cyts-c, only one Trp residue is present in whole protein sequence. As suggested by X-ray crystallography [111, 112, 220–223], the side chain of Trp59 (indole N1) is H-bonded to the heme propionate, acquires a portion of the heme crevice, and provides a unique electronic/hydrophobic environment to the heme crevice. Various variants of Trp59 have been synthesized and all of them showed a reduced biological function [28, 210]. It is the largest naturally occurring amino acid, hydrophobic, planar in structure, and aromatic. This unique physical characteristic of Trp made it conserved at position 59. Moreover, Trp59 fluorescence is extremely sensitive to the overall protein conformation of cyt-c [224]. Enormous work has already been carried out to analyze the role and importance of the Trp in cyt-c.
Mutants with Ser, Cys, and Gly at position 59 were found to be thermolabile [210] and non-functional [223, 225]. Trp59His was also non-functional as reported by Lett et al. [226]. Formylation of Trp59 resulted in a significant decrease in enzymatic activity (succinate oxidase activity) of cyt-c along with the disruption of 695-nm band [227]. Though Trp59Phe showed growth rates as good as WT, it showed a decrease of 37 mV in the redox potential [226]. Mutant Trp59Tyr and Trp59Leu also retained a reasonable level of function [28, 210]. Substitution of highly conserved Trp67 (Rhodobacter capsulatus cytochrome c2), an analogue to Trp59 of h-cyt-c, with Tyr resulted in significant decrease in conformational stability (ΔΔG D = −2.5 kcal/mol) while the Met80–heme bond was destabilized by 1 kcal/mol along with an alteration in heme environment (red shift in absorbance maxima) [228] (Table 2). The order in which the stability and specific activity decreased for the different mutants at position 59 is Phe> Tyr> Leu> Ser> Cys> Gly. Stability analyzed here was a qualitative assessment of yield and color of the isolated proteins [210]. It should be noted that, if Trp59 was substituted with hydrophobic residues (such as Phe, Tyr) mutants retained some function, whereas substitution with Gly or Ser caused complete inactivation. These observations suggest that hydrophobic interactions of Trp59 with inner propionic acid play an important role in stability of cyt-c [210].
Aviram and Schejter [227] suggested that the hydrophobicity and H-bond that is present in between its indole moiety and propionate group of the heme are important in stabilizing the protein structure possibly by modulating the Met80–heme–Fe coordination geometry or by a hydrogen-bonding interaction with the heme moiety. Black et al. [229] analyzed individual side-chain features of Trp by substituting it with three non-coded semi synthetic analogues: p-iodophenylalanine (IPA), β-(3-pyridyl)-alanine (BPA) or β-(2-naphthyl)-alanine (BNA), because they vary from Trp with respect to size, hydrophobicity and H-bond potential. BNA resembled Trp most closely in size and planarity but it lacked the electropositive region associated with the indole imine. Similarly, IPA is similar in size but neither it is planar nor it can form H-bond. On the other hand, BPA was much smaller and nonpolar but was able to act as H-bond donor. Redox potential measurements and succinate oxidase assay showed a significant decrease in activity (21 mV and 60 ± 1 % activity, respectively) for BPA as compared to other two variants in reference to the control (Lys55Met). This result attributed a more important role to bulky aromatic side chain of Trp than its H-bonding ability in maintaining the redox potential as well as electron transfer process of the protein. On the other hand, cytochrome oxidase (COX) assay resulted in marked reduction in activity for all the three variants, suggesting that loss of any characteristics of Trp lead to defective tunneling of electrons between the redox centers [229]. It can be then concluded that hydrophobic, planar, and aromatic character of Trp side chain has a profound impact on global stability and in maintaining the electrostatic environment around heme.
Tyr67
Tyr67 is a well-conserved residue located within the helix (residues 59–69) with few exceptions (Table 1). The hydroxyl group of Tyr67 modulates spectral properties of the heme and has intense influence on its redox properties [230]. The H-bonding patterns are thought to differ in oxidized and reduced proteins. In its reduced state, the side chain hydroxyl group of Tyr67 forms extensive H-bonding network with side chains of Asn52, Thr78, and Met80 [112, 192] and the internally bound conserved water molecule, Wat166 [111, 231]. While in the oxidized cyt-c, reorientation of the Wat166 disrupts all these H-bonds [192]. Such a change in hydrogen bonding pattern and electrostatic shift of Wat166 are thought to govern various oxidation state-dependent conformational changes [192] as well as modulate the redox potential and stability cyt-c [209, 230, 232–234]. Tyr67 supposedly monitors the polarity and number of water molecule in the heme cavity and these factors are important determinants of redox potential [235].
Tyr67Phe is the most studied mutant [230, 233, 236, 237]. In this mutant, besides Wat166, there is an additional water molecule (Wat300) in the place occupied by the hydroxyl group of Tyr side chain in WT protein. In the reduced protein, Wat300 maintains the hydrogen bond network [238]. On the contrary, the two water molecules realign and disrupt H-bond network leading to destabilization of oxydized mutant Tyr67Phe [238]. Thus, the stability of the alternative oxidation states differs, with coincident modification in the local flexibility of nearby polypeptide chain (residue 65–72) [238]. In Tyr67Phe of rat cyt-c, the mutation led to an increase in the local stability with an increase in T m (midpoint of denaturation) from 60 to 90 °C with a simultaneous decrease in the ∆G D of WT = 9.15 kcal/mol while ∆G D of Y67F mutant = 7.19 kcal/mol [98] (Table 2). A similar trend was not observed for the yeast mutant [98, 230, 237, 238]. Tyr67Phe mutant is more stable (T m = 107 °C) in terms of T m than the WT (T m = 65 °C).
Soret spectrum of Tyr67Phe mutant of h-cyt-c showed a 3-nm shift in the α- and β-bands [230], which indicate a modified electronic heme structure [238]. Wallace et al. [230] inferred this to be due to the absence of hydroxyl group of Tyr, which modified the polarity and abolished H-bonding capability of the side chain shifting the electron distribution in the π-cloud of the porphyrin ring [239] and thus led to the red shift. Moreover, the hydroxyl group of tyrosine blocks the access of the molecular oxygen thus inhibiting the auto-oxidation of ferrocyt-c [230].
Another important effect that appeared to be due to this mutation was a sudden drop in the midpoint reduction potential, 35 mV drop in the horse heart Tyr67Phe mutant [230], ~25–27 mV drop for the rat Tyr67Phe mutant [240], and ~56–60 mV for Tyr67Phe of iso-1-cyt-c [238, 240]. Such a decrease in redox potential contradicts the conventional theories [100, 241], which suggest that the reduction potential should increase as hydrophobicity of the heme pocket rises, which is expected in case of Tyr67Phe. Wallace et al. [230] attributed this variation of potential due to the dramatic change in electronic structure of the heme group induced by the presence or absence of the phenolic hydroxyl group and signaled by the red shift in major absorption bands as discussed above. Berghuis et al. [238] provided an alternative mechanism for such behavior. As reported earlier [2, 242], electron affinity of Met80 ligand is considered to be the major contributor in determining the reduction potential of cyt-c. Thus, according to Berghuis et al. [192, 238], the electron affinity of Met80 ligand would be affected by the presence or absence of the hydrogen bond between Tyr67-OH and Met80-SD, thus contributing to the value of the midpoint reduction potential of the protein. So, in case of Tyr67Phe, removal of this bond supposedly leads to a decrease in the reduction potential [238].
Ying et al. [13] expressed Tyr67His and Tyr67Arg variants, and observed that the H-bond network around Tyr67 is associated with the conformational transition of cyt-c, such a conformational intermediate in cyt-c has high peroxidase activity, which suggests that the H-bond network around Tyr67 is essential in maintaining the cyt-c functioning as an electron transfer protein as well as a trigger in apoptosis. Tyr67 does not participate in electron transfer as suggested in earlier studies [230, 243]. A conclusion of our analysis is that the hydroxyl group has a specific function in setting the redox potential, probably by influencing electron distribution in the heme and may inhibit autoxidation of ferrocyt-c by blocking access of oxygen. Recently, Battistuzzi et al. [171] have shown that H-bonds associated with Tyr67 connect Ω loops (40–57 and 71–85), which are involved in pH-dependent denaturation. Thus, disruption of this H-bonding network (as in Tyr67Ala) would lead to disruption of the tertiary structure, inducing molten globule conformation in acid-induced unfolding. From the above discussion, we may also conclude that the natural residue at position 67, Tyr, is also evolutionarily conserved for a role in structural stabilization.
Phe82
Phe82 is a phylogenetically conserved residue of mitochondrial cyts-c [2, 129]. It is located on the surface region of the protein and forms complex interactions with redox partners [112]. It is also in close proximity to the heme and thus attributed to important functional roles [244]. The most important roles that it performs are maintaining the redox potential of the protein and influencing the mechanisms of electron transfer and complex formation of cyt-c with its redox partners [245–253]. Thus, any mutation at this position leads to alteration in stability (Table 2), and electrochemical and kinetic properties of the protein [251].
Growth rate analysis of different mutants with all the other 19 amino acids at position 82 has shown that the mutants (except for Phe82Cys) were successfully able to support respiration and growth on non-fermentable carbon source, indicating that mutant proteins can reduce COX [254, 255]. Such studies suggest that cyt-c can tolerate a wide range of amino acid replacements at this position and can survive. However, the inability of mutant with stop codon at this position to grow and significantly reduced growth in Phe82Cys variant [254] has confirmed the absolute conservation of Phe82. Replacement with Pro and Lys also showed a much reduced growth rate, probably due to some structural or electrochemical perturbation in the heme pocket [254].
Various structural and functional studies have revealed that the residue Phe82 has a very important role to play in maintaining the functional properties of the protein. Various experimental studies [245, 256, 257] as well as molecular dynamics simulations [258] with position-82 mutants have revealed crucial roles for this residue in regulating the rate of electron transfer from y-ferrocyt-c and affects the alkaline isomerization and thus influences the heme–ligand interaction [259]. This residue also stabilizes the native heme environment [248, 249, 259]. Any mutation at this position reportedly affects the redox potential, reduction rate, and spectroscopic properties (implying structural perturbation) of the protein [251].
Electrochemical, kinetic, and spectroscopic properties of variants at position 82 have been extensively studied with three-dimensional structures known for a few of the mutants [248, 249] along with the WT cyt-c [111, 149, 220, 260]. Studies have revealed that the side chain of Phe at this position maintains the overall conformation of the protein and adds to the non-polar nature of the heme environment [247–249, 251]. The almost-coplanar position of Phe82 with respect to heme [220] allows its nonpolar side chain to effectively exclude the solvent access to the quadrant of heme pocket in which it is positioned [248].
Rafferty et al. [251] observed a direct correlation of the size of the residue at position 82 with the reduction potentials in the various mutants they expressed at pH 6.0. The potential progressively lowers as the size of the residue at this position decreases in the order Phe> Leu> Tyr> Ile> Ala> Ser> Gly. Solvent accessibility is an important factor in deciding the reduction potential; an example could be seen in case of Phe82 variants. The surface area of heme exposed to the external solvent in WT protein increases from 9.6 % [220] to 13.7 % (of the total heme surface) in Phe82Ser mutant [249]. The mutant simultaneously shows a fall of 35 mV in its reduction potential as compared to the WT [249]. Thus, increased accessibility and resultant polarization led to a fall in the redox potential of the protein. On the contrary, Phe82Gly, with almost similar heme exposure (8.7 %) as in WT, also showed a drop in redox potential [248]. Louie and Brayer [248] attributed this drop to the absence of side chain and consequent refolding, which prevented an increase in solvent accessibility but allowed the polar groups around to enter the heme pocket and thus led to a fall in the reduction potential. In case of Phe82Tyr, the increased polarity associated with the tyrosyl side chain was supposed to have a larger effect, but a slight change was observed in heme reduction potential [251]. Lo et al. [247] suggested this mild effect to be due to a small positional shift in this group, which orients out the tyrosyl hydroxyl group toward the protein surface where it can interact with solvent molecules instead of altering the polarity of the heme pocket.
A profound effect has been observed on electron transfer rates when the aromatic side chain of Phe82 was replaced with an aliphatic side chain even when it is of comparable size [256]. Structural, complexation, and electron transfer studies of cyt-c and its redox partners have revealed that the delocalized π-electron system of the heme is coupled with that of aromatic side chain of Phe, providing a potential passage for the electron transfer between cyt-c and its redox partners [245, 257, 261]. Electron transfer rates as studied between reduced cyt-c and Zn substituted cyt-c peroxidase supported the above observation, as rates with Tyr or Phe at this position were 104 times higher than those of mutants with aliphatic side chains [245]. While in the case of Phe82Gly, the absence of side chain in Gly leads to major polypeptide rearrangement, altering the interacting domains and making complex formation of cyt-c with its redox partners inefficient. Hence, the electron transfer activity is reduced [248]. The absence of a side chain also renders Gly, at position 82, unable to interact with π-electrons of the heme group, thus drastically affecting the electron transfer activity of cyt-c [248]. Other evidence to prove the fact that the nature of residue at position 82 is important comes from the result of kinetic study [255]; different variants of this position exhibited different steady-state kinetics. The mutant Phe82Ser, which has lesser redox potential than that of the WT, was supposed to facilitate the transfer rate and raise the steady-state activity with cyt-c peroxidase. However, the results were just the opposite, suggesting structural perturbation, which most likely accounts for the decreased on-kinetics and/or off-kinetics [255]. Thus, all the above studies show that the aromatic character of Phe along with the structural perturbation induced due to mutation is important in modulating electron transfer rates.
The absence of the side chain in Phe82Gly led to relocation of Wat166, loss of H-bond with Asn52, and remarkable but restricted refolding of the polypeptide chain around the mutation site [248], whereas the presence of the hydroxyl group in Phe82Tyr mutant increased the thermal mobility, making the protein less stable [262]. Lum et al. [263] have shown that hydrophobic and hydrophilic interactions formed in the region around Phe82 is critically important to the formation of y-cyt-c and cyt-c-peroxidase complex.
Prolines
Prolines are found to be highly conserved at positions 30, 71, and 76. A unique cyclic structure and the associated conformational rigidity of proline contribute to the conformational integrity of the cyt-c structure, a motif common to both mitochondrial and the more variable prokaryotic cyts-c [264]. Though its side chain offers no distinctive chemical attributes, the complex folding/unfolding kinetics of many proteins (ribonuclease, cyt-c, metmyoglobin) [265, 266] is found associated with cis–trans isomerism of proline residues [267–269]. Here, we are going to discuss the role of three conserved proline residues. Their conservation among mitochondrial cyts-c indicates that they are important for proper folding and function. Pro30 is buried in the protein interior between two sharp bends essentially important in maintaining the orientation of the heme ligand, His18, whereas Pro71 is located in between helix III (residue 60–70) and IV (residues 70–75) [149], acting as a helix breaker. On the other hand, Pro76 is present as a second residue of a type II β-turn, and found to be partially exposed on the protein surface [111, 112].
Sequence analysis shows that Pro30 is highly conserved in all cyts-c. Pro30 follows a sharp γ bend of the chain, defined by the Lys27, Thr/Val28, and the invariant Gly29 [270]. The residues 26–30, a segment of structure, is relatively rigid due to the woven network of H-bonds [270]. Pro30 is important in holding His18 to its location in the heme crevice [28]. It probably contributes in fixing the imidazole ring [149] at its position with heme and forms the closed-crevice [271] structure of cyt-c [111, 112, 220, 249, 272]. Substitution of Pro with smaller residues (Ala, Val) leads to creation of a cavity in a highly hydrophobic environment thus dramatically decreasing the stability of Met80–Fe bond [273].
Replacement of Pro30 with Leu or Thr showed either very poor aerobic growth or almost no growth, presumably due to the inefficient maturation or instability, rendering the protein functionally incompetent to support the normal growth of the mutant organism [28, 274, 275]. Poerio et al. [274] have also shown a drastic decrease in the activity of the protein with Gly at this position. However Pro30 to Gly30 change is less destabilizing than replacement of other less highly conserved residues. Instability and poor in vivo function for position 30 mutants are in concurrence with sequence analysis that suggests a strong preference for Pro at this position. Cyt-c 2 of Rhodobacter capsulatus represents a natural variant of corresponding Pro to Ala [276]. Furthermore, Koshy et al. [270] suggested that substitution of Pro30 by Ala/Val causes drastic alteration in the stability of the heme–Met80 bond without much affect on the conformational stability of the protein.
Another structurally significant residue is Pro71 located at the intersection of helix III and IV forming H-bond to Ile75. It lies opposite to Tyr67, Met80, and Phe82 in such a way that its main chain and side chain atoms are occluded from bulk solvent forming a hydrophobic patch [277]. It helps in excluding solvent from the heme pocket. The size and the chemical nature of the side chain to be accommodated makes proline to be a preferential choice at this typical position. Alteration in activity and protein folding on replacement with bulkier or hydrophilic residues suggest its significance in maintaining the functionality and directing the folding process of cyt-c in this region [149, 220, 278]. Due to the flexibility in the region Pro71 to Gly83 of polypeptide in the oxidized cyt-c, it was suggested to play functional roles in oxidation state-dependent conformational changes [192]. Detailed analysis of amino acid preferences at the ends of α-helices also strongly suggest that proline is most preferred at C-cap+ 1 and quiet preferred at N-cap+ 1 (2.6:1 preference over other residues) which is in perfect accordance with its position 71 [200, 279]. The conformational rigidity that it provides at position 71 induces a right angle between the two helices restricting the movement of the polypeptide in this region thus positioning Met80 in order to properly co-ordinate with heme [279].
Various studies with substitution mutation have been carried out to further understand the role of Pro71 by replacing structurally and chemically unique side chains with different bulkier and/or charged residues [28, 268, 275, 277–282]. Mutation altering the codon for proline to a nonsense codon (cyc1-11 strain) led to absolute deficiency of y-cyt-c. Unlike UAA mutations and other cyc1 (gene coding for iso-1-cyt-c) mutations that revert by single base pair substitution, reversion studies carried out with the use of various mutagens showed lowest reversion rate for cyc1-11 strain. Functional revertants showed decreased activity (Pro71> Val71> Thr71> Ser71> Ile71). Absence of other residues (Glx, Lys, Tyr, Leu) in revertants at this position by single base pair substitution was interpreted to be producing non-functional proteins justifying their absence [278]. In vitro stability analysis (∆G D estimation by guanidine hydrochloride induced equilibrium studies) of the mutants (Val71, Thr71, Ile71) also showed to follow the same decreasing trend (Table 2) as was obtained by Ernst et al. [281] for the in vivo activity.
X-ray structures of Pro71 mutants (Pro71Val, Pro71Thr, and Pro71Ile) showed some displacement in the polypeptide region that secures the heme crevice from solvent accessibility [283]. Nva71 (norvalin is a semi-synthesized amino acid) exhibited complete loss of the 695-nm band, decline in ATP affinity, change in dipole moment, decrease in redox potential, fall in alkaline pK and relative destabilization, and thus causing significant loss of electron transfer ability [279]. These observations affirm that Pro71 has a significant role in maintaining axial coordination and function of cyt-c [2, 153, 284]. Nevertheless, the dominant factor for the loss of function results from the primary role of proline in making the β turn that defines the optimal conformation of the 70-s loop for correct heme iron coordination. Absence of any phylogenetic variability also supports its conservation. Another mutant, Pro71His, showed a difference in axial coordination with simultaneous oxidation state-dependent conformational change [277]. In case of oxidized Pro71His, Fe(III) (stronger Lewis acid) binds to side chain imidazole ring of His71 (a stronger Lewis base) as its axial ligand simultaneously pushing Met80 away as judged by the absence of 695-nm band retaining the hydrophobicity as was present in the WT molecule. In case of reduced Pro71His, Fe(II) (weaker Lewis acid) associates with the side chain of Met80. Such wide-ranging conformational changes associated with different oxidation states in Pro71His exemplifies the ability of cyt-c to deal with extreme situations of variation in pH, temperature, etc., while still retaining its stability and function. Although there is no change in secondary structure, the flexibility that it attains due to substitution of Pro at this position leads to switching between axial ligands thus resulting in conformational variance [277]. Furthermore, the conserved residue Pro71 is suggested to act as a helix breaker in the region of residues 60–74, and it functions as a key hydrophobic patch to prevent the solvent accessibility to heme [277]. It can also be concluded that Pro71 provides the conformational rigidity to maintain the desirable Met coordination.
Pro76 is a highly conserved residue and is a part of loop D (residues 70–84), which has been an extremely conserved segment throughout the evolution unlike other surface loops [285]. Pro76 along with Gly77 forms the β turn, which orients the peptide backbone into loop, which facilitates proper coordination of Fe-Met80 coordination and provides a complementary surface to physiological partners [286]. Substitution with Ala at position 76 significantly altered the dihedral angles within the loop, thus withdrawing its positional constraints affecting heme–Met80 coordination, indirectly altering the physicochemical and biological activities of cyt-c [286]. Substitution with different kinds of residues (Leu/Gly/Arg/Ser/Tyr/Trp) at position 76 showed, except for Pro76Val, a drastic drop in functional capacity of the mutant protein as well as the amount of holoprotein [285, 287]. Thermal stability of all the mutants (Leu/Gly/Val/Arg/Sr/Tyr/Trp substituted at position 76) showed an almost constant decrease in T m (value in the range 47–48 °C) with respect to the WT (T m = 53 °C) (Table 2). These observations suggest that the presence of bulkier (Pro76Trp) or lighter (Pro76Gly) residues at this position did not make any difference in the thermal stability [287]. It is likely that mutation of the imino acid proline at position 76 to an amino acid has increased the entropy of the denatured state, resulting in an apparent decrease in the stability of the native state [287].
Other conserved residues
Lys72
Lys72 is located in the highly conserved region and is important for binding to redox partner’s COX and cyt-c bc 1 complex [1, 288–291]. In contrast to higher animals, methylation of Lys72 in fungi and plants has been a point of speculation. The biological function and importance of cyt-c methylation have not been clearly established [292–294]. Various studies involving mutant forms of y-cyt-c lacking trimethylated Lys72 showed to preserve nearly full activity in vivo [294, 295]. Moreover, in higher animals, which have unmethylated Lys72, this residue plays a significant role in the apoptogenic activity of cyt-c [7, 296].
The function of trimethylated Lys72 has been investigated in yeast in various studies [297–301]. Mutant Lys72Arg (unmethylated cyt-c) demonstrated a minimal change in conformation and stability as well as indistinguishable growth patterns in lactate medium as compared to the WT protein, indicating normal in vivo activities [295]. A slight decrease in binding of cyt-c to cyt-b 2 concurred the previous suggestions [302, 303] that (CH3)3,-Lys-72 may play a specific role in recognition and binding of the physiological partners of cyt-c [295]. Several studies have shown that methylation of the Lys72 of apocyt-c increases its import into mitochondria preferentially into yeast, but not rat liver [293, 304]. The reasons provided for this preference [293] were that methylation of apocyt-c dramatically lowers its isoelectric point (against a predicted increase) and decreases its Stokes radius. Frost et al. [304] have shown that the methylation state of the apoprotein has no significant effect on its conversion to holoprotein, suggesting that the import mechanism is separate from the heme-attaching activity. They also showed that methylation of apocyt-c alters the conformation of the whole protein. Fully extended conformation of trimethyllysine in y-cyt-c and the steric obstruction of the methyl groups is suggested to interfere in apoptosome formation, thus preventing induction of apoptosis [297].
Cyt-c, in mitochondrial inter-membrane space, is an important factor in the electron transport chain [1], but when translocated to cytosol, it plays an important role in the early stages of programmed cell death [4, 7]. Cyt-c, on stimulation for apoptosis, cyt-c gets released in cytosol to bind with Apaf-I (apoptotic protease-activating factor) to form apoptosome, which converts pro-caspase 9 to active caspase 9, eventually activating the downstream effectors of apoptosis [305–307]. Lys72 is one of the few residues that were found to be important for cyt-c-Apaf-I interaction [308, 309].
To establish the contribution of Lys72 in apoptogenic activity of cyt-c, Chertkova et al. [308] produced h-cyt-c mutants with various substitutions at position 72. Their cell-based results were in agreement with results reported in previous studies [5] for cell-free (Xenopus egg/Mammalian cell extracts) systems, i.e., a significant drop in the apoptotic activity of the mutants. The order for substituted residues in which the activity declined in mutants was found to be different for in vitro (Arg> Gly> Ala> Glu> Leu) and in vivo (Ala> Leu> Glu) conditions [296]. Whereas Lys72Trp substitution in horse as well as murine cyt-c caused absolute loss of apoptogenic activity, other properties (electron transport) of mutant protein remain unperturbed [5, 296, 297]. Lys72Trp represents one of the very few examples of a mammalian mutant cyt-c (except trimethylated cyt-c) where single substitution leads to absolute loss of apoptogenic activity [12, 308].
Cyt-c in the inter-membrane space could be either loosely bound or tightly bound to the membrane. The loosely bound cyts-c, which could undergo frequent association/dissociation, are suitable for a normal redox cycle, whereas the tightly bound ones are suggested to be involved in triggering the apoptotic cycle. For the association of the cyt-c to the membrane, electrostatic interaction between the two is the prerequisite followed by conformational changes and loosening of the tertiary structure of cyt-c [310–316]. Lys72 is one of the few basic residues that complements the negatively charged membrane surface and thus is essential for the anchorage of membrane surface in cyt-c [3].
Thr78
Thr78 is another conserved residue with few exceptions (Table 1). It is involved in a network of H-bonds around heme. Its side chain is packed against the heme, and plays a significant role in forming the heme pocket and maintaining the heme environment [220]. Wallace et al. [230] synthesized three analogues Thr78Val, Thr78Aba (aminobutyric acid), and Thr78Asn and carried out absorption measurements. It was observed that these analogues give different results. Thr78Val completely lacked the 695-nm band, which is supposed to be the consequence of shifts in pK and T m. The Thr78Aba analogue showed a very weak band at 695 nm, suggesting the importance of the single H-bond provided by the Thr. Thr78Asn is destabilized, and, as suggested by Wallace et al. [230], is due to the higher entropic cost of burying the side chain of Asn compared to that of Thr. However, the presence of Asn in a few of the naturally occurring forms contradicts this argument. Dickerson and Timkovich [317] explained these contradicting observations on the basis of different stability for the natural variants living in different temperature conditions. They showed that Thr78Asn at 37 °C is more than 50 % in the unproductive state IV in which the iron-sulfur co-ordination is lost, and at 10 °C it is >90 % in the normal state III. Such natural variants of cyt-c would be inefficient in animal species at an elevated body temperature, and would be much less so in species living in cold water, such as Chlamydomonas. Alternatively, this organism might possess a change elsewhere in the sequence that can compensate for the Thr to Asn mutation.
Under standard conditions (pH 7.0, 25 °C) the analogue Thr78Asn has a normal redox potential, whereas Thr78Aba showed a sharp drop (~35 mV). At pH 6.0, it (Thr78Aba) showed to completely restore normalcy. Therefore, it was suggested that the destabilization of the heme crevice causes the redox potential change due to increased solvent accessibility, or the change in ligation at the sixth coordination position. Thr78 is essentially involved in important stabilizing interaction [230]. The requirement is highly specific and the anomalously low biological activity of the analogues prepared suggests that this residue has a direct role in electron transfer [230].
Post-translational modifications
Besides methylation (discussed in “Lys72”), there are some other post-translational modifications such as phosphorylation, nitration for cyt-c. Reversible phosphorylation is one of the methods to regulate important metabolic enzymes. Huttemann et al. [12, 205, 318] have shown mammalian cyt-c to be tyrosine phosphorylated at two novel sites, namely, Tyr48 (cow liver cyt-c) and Tyr97 (cow heart cyt-c) [12, 205, 318]. Thr28 and Ser47 are two other phosphorylation sites that have been reported in cyt-c of human skeletal muscle. The presence of phosphotyrosines at distinct positions in different tissues suggests that cyt-c is an active member in regulatory networks and is being regulated by a cell signaling network in a tissue-specific manner. Phosphorylation has shown to affect electron transfer properties as well as apoptosis-inducing ability of cyt-c. Phosphorylated Tyr97 is proposed to form a salt bridge with adjacently lying positively charged Lys7 (important residue in apoptosome formation) [309], thus preventing induction of apoptosis [205]. Tyr97 phosphorylation has also shown to enhance sigmoidal kinetics with COX and inhibit its reaction with COX [205]. On the other hand, Tyr48 phosphorylation has shown to cause a 50 % reduction in cyt-c oxidation or respiration [318]. Further characterization of the role of phosphorylation has been reported with the Tyr48Glu mutant of cyt-c, which mimics in vivo Tyr48 phosphorylation. The presence of negative charge either in the form of Glu48 or phosphate group attached to Tyr 48 decreases the binding affinity of cardiolipin to cyt-c, a major preapoptotic event, thus interfering in the induction of apoptosis. Owing to the negative charge, perturbation in the heme pocket also caused a fall in redox potential in Tyr48Glu [12]. Pecina et al. [12] has suggested that alleviating cyt-c’s role in respiratory chain may favor its other functions as a peroxidase or antioxidant. Tyr48Glu also rendered cyt-c unable to induce caspase-3 activation in cytoplasmic extracts thus again inhibiting apoptosis [12]. Functional relevance of phosphorylation of Thr28 and Ser47 has not yet been elucidated. Thus, it can be concluded that phosphorylation in eukaryotic cyt-c regulates all aspects of cyt-c function, ranging from electron transport to peroxidase activity to apoptosis induction.
In vivo tyrosine nitration, which is another post-translational modification, occurs by the addition of a –NO2 group at the ortho-position of the phenolic hydroxyl group. It is apparently a more stable and specific modification as compared to tyrosine phosphorylation [319–321]. Implications of nitration on functional and physicochemical aspects of cyt-c have been widely studied [9, 321–329]. Nitration of different Tyr residues produces different alterations in cyt-c function [9, 327, 330]. Radi et al. [321–323, 328, 331, 332] have found a range of biologically significant reaction mechanisms that may carry out Tyr nitration in cyt-c involving various reactive nitrogen species (RNS e.g., nitric oxide and its derived oxidants). Nitration of cyt-c prevents the electron transport [323, 324, 327, 333], disrupts mitochondrial membrane potential and considerably enhances the peroxidase activity of cyt-c [323, 334]. Cyt-c being hexa-coordinated in its native state show weak peroxidase activity, whereas penta-coordinated state of heme is the characteristic feature of peroxidases [335–337]. During unfolding due to binding with anionic lipids or chemical modifications (such as carboxymethylation or nitration), cyt-c attains this conformation with disruption of Met80–heme interaction [11, 323, 338]. Peroxidase activity depends on the rate of exchange of Fe axial ligand by H2O2 [9]. Out of all Tyr residues in cyt-c, only three, namely, Tyr67, Tyr74, and Tyr97, are found to be nitrated in vivo [321, 322, 339]. Solvent-exposed Tyr74 and Tyr97 are the preferred targets of peroxynitrite-mediated nitration of cyt-c [322, 340]. Peroxynitrite is a strong oxidant/RNS, which is an in vivo nitrating agent [341, 342]. In vivo nitration of Tyr67 has also been reported and is supposedly favored due to the proximity of the heme group [321, 339]. Though well accessible to solvent, Tyr46 (present in many eukaryotes) and Tyr48 have not yet been reported to be nitrated in vivo. However, the effect of nitration of these residues has been studied in vitro [330]. Nitration of Tyr74 affects the dynamics of Ω loop (residues 70–85) containing Met80–heme coordination bond and makes the heme moiety accessible to H2O2, leading to alteration in the peroxidase activity of the protein. Nitration of Tyr74 also induces early alkaline transition, shifting pKa towards neutral pH and renders cyt-c unable to react with COX [9, 340]. Shifting of alkaline transition towards physiological pH replaces Met80 by Lys73 or Lys79 as heme axial ligands (also present in the Ω loop). Such conformational changes alter the electrostatic properties of the protein surface that lead to the formation of a non-functional apoptosome, making cyt-c unable to activate caspases and to induce apoptosis [9]. Therefore, the post-translationally modified membrane anchored cyt-c upon nitration (esp. Tyr74), essentially triggers the early apoptotic signaling events, whereas the cytoplasmic non-nitrated cyt-c amplifies the apoptotic signal by caspases cascade activation [9]. It should be noted that Tyr97 and Tyr67 also enhance the peroxidase activity upon nitration but less significantly than that of nitrated Tyr74 [9, 323, 336, 340].
Another post-translational modification that still needs to be focused with reference to its functional relevance is acetylation. Kim et al. [343] suggested that ~20 % of mitochondrial proteins are lysine acetylated and have shown that cyt-c also has lysine acetylation site. Lysine acetylation, besides neutralizing Lys’s positive charge, also increases its hydrophobicity and side chain length, thus inducing alteration in protein conformation, probably affecting functional property of the protein. Chemically acetylated cyt-c has shown to drastically affect the redox properties of the protein [344]. Though in vivo lysine acetylation in cyt-c is not a well-studied phenomenon, but with the analysis of the other identified acetylated proteins, Kim et al. [343] suggested its role in altering protein–protein interactions, enzymatic activity, and other lysine modifications such as methylation. On the other hand, N-terminal acetylation, a co-translational modification, is a well-studied phenomenon occurring in most (~85 %) of eukaryotic proteins including cyt-c [345–347]. It involves transfer of acetyl group from acetyl-CoA to the termini of α amino group by N- terminal acetyl transferases. In cyt-c, N-terminal acetylation has shown to prevent degradation in vitro [348, 349].
Isoforms of cyt-c
Besides post-translational modifications, cyt-c functions are also found to be regulated by tissue-specific isoforms of the protein. Different isoforms for cyt-c have been reported for various species, suggesting a requirement for specialized polypeptide functions or regulatory necessities that cannot be served by a single gene. For example, in case of S. cerevisiae, there are two isoforms, namely, iso-1- and iso-2-cyt-c, which are encoded by two differently regulated genes and have adapted different structural characteristics in response to nutritional variation [132, 350, 351]. On the other hand, isoforms of cyt-c in Drosophila (cyt-cp and cyt-cd) are tissue-specific, and their genes show developmental regulation in their pattern of expression [352–354]. Furthermore, a unique isoform of cyt-c specific for testicular cells (cyt-c t), other than the normal cyt-c present in somatic cells (cyt-c s) in various mammals such as rodents, rabbit, bull, etc., have been reported [355–358]. Moreover, a pseudogene similar to cyt-c t in human genome and similar coding DNA sequence from cDNA of cow has also been reported (GenBank accession no. AAI02715.1) [352]. Non-transcribed pseudogene in humans is suggested to be the cyt-c t, long lost during primate evolution [352, 359]. Thus, with all these pieces of evidence, Liu et al. [14] elucidate, whether functional or not, that the cyt-c t gene is prevalent in mammals.
Isoforms of cyt-c in rodents are very well studied proteins. Cyt-c t, expressed in mammalian male germ cells, are different from their somatic counterparts in their expression pattern as well as in intron–exon genomic organization, length of the 5′ untranslated region, and number of polyadenylation sites [360, 361]. As reported by Hennig et al. [362] and Huttemann et al. [36], most of the substitutions in cyt-c t in mouse with reference to cyt-c s are located on one side of the protein, suggesting a functional relevance of such substitutions. Crystal structure analysis also suggests an alteration in water pattern and environment around Arg38 (a residue important in maintaining the redox behavior and ascorbate reduction of cyt-c [2, 247, 363]) and in cyt-c t when compared to cyt-c s. More specific requirements of antioxidation and apoptosis in sperms justify the evolution of tissue (testis)-specific protein, cyt-c t. High apoptotic activity (3–5 times higher than that of cyt-c s) of cyt-c t is supposed to be important in regulating the quantity and quality of sperm cells by undergoing apoptosis comparatively easily in reacting to reactive oxygen species (ROS) [14, 360]. ROS, generated as a result of electron transport reactions taking place in mitochondria, are subsequently released in matrix and intermembrane space by complex I and III, respectively [364–368]. These ROS may damage DNA and other cellular structures and, if uncontrolled, may lead to apoptosis [369]. Thus cyt-c t, which displays a threefold higher capacity to destroy ROS, esp. H2O2, is essential to downregulate higher ROS production, which is a consequence of high energy production in sperms and thus ensuring integrity of the genome transmitted by the sperm [14]. The existence of different isoforms of cyt-c suggests that conservation along with various changes induced (amino acid substitution or conformational change) in a protein during the course of evolution is in accordance with the specific requirements (functional, regulatory, etc.).
Concluding remarks
Cyt-c, an indispensable member of the electron transport chain, is vital for maintaining cell life and apoptosis. Functions of cyt-c are governed by its hydrophobic core composed of covalently bound heme along with its axial ligands and other H-bonded residues. Oxidation state, polarity, and conformational changes in heme crevice provide cyt-c its requisite function. The residues, which are critical in maintaining these features of cyt-c, are made conserved by evolution. Any mutation in these key residues leads to dramatic alteration in structure, function, and stability of the protein.
Alteration in a few residues, such as Cys14/17 and His18, abolish heme attachment, rendering the protein non-functional. On the other hand, some residues (e.g., Gly6, Phe10, Phe82) allow broad range of substitutions at their positions but significantly affect the structure, function, and stability of the protein. Residues that bind heme covalently to the polypeptide are Cys14/17, making cyt-c uniquely different from other cyts. His18 is a tightly bound fifth-axial ligand, stabilizing the heme along with significantly contributing to the functional and structural aspects of cyt-c. On the other hand, Met80, the sixth-axial ligand, is highly labile but very crucial in maintaining redox potential of the protein, andthus directly affects the electron transfer activity of cyt-c.
Interface formed by the perpendicular pairing of N-terminal (residues 6–14) and C-terminal (residues 87–102) helices is the most conserved structural motif in all cyts-c and represents an early event in cyt-c folding. The residues of these helices interact in a specific manner. Absence of the side chain in Gly6 allows the side chain of Leu94 to fit in, whereas, Phe10 and Tyr97 share protein stabilizing aromatic–aromatic interaction. Leu94 have a role in the formation of early and late intermediates along the folding pathway of cyt-c.
Trp59, mostly single of its kind in the whole sequence of cyt-c, is highly conserved due to its unique aromatic ring structure. Replacements with natural as well as semi-synthetic analogues showed a prominent role of Trp59 in structure, stability, electron transport, and activity of the protein. Tyr67, another conserved aromatic residue, is involved in extensive H-bonding network in the heme crevice, thus, imperative in maintaining the redox potential of the protein. It also contributes to the pro-apoptotic activity of cyt-c. Phe82, besides being on the surface of the protein, is also in proximity to heme, and is important for electron transfer and coupling between the cyt-c and its redox partners. Its aromatic side chain also contributes to the hydrophobic environment of the heme crevice.
Prolines are highly conserved, contributing to the conformational integrity of cyt-c by virtue of their unique cyclic structure. Pro30/71 residues contribute in maintaining the proper axial ligation of heme, and thus indirectly contribute to structure and function of cyt-c. Pro76 also contributes to structural stabilization. Lys72 is located on the surface region important for binding to redox partners, thus it has an important role in electron transport. It is also important in anchorage of cyt-c to negatively charged membrane and triggering of apoptosis. Thr78, on the other hand, is located closer to the heme and is involved in intensive H-bonding network around heme. It has a significant contribution in maintaining the heme environment.
Besides conserving the residues which preserve the electron transfer property of cyt-c, residues contributing to, apoptosis, yet another very important function of the protein, are also made conserved. These residues, Tyr48 (highly conserved in eukaryotes), Tyr67, Tyr74 (conserved in many eukaryotes), Lys72 and Tyr97, are post-translationally regulated. Any kind of alteration in these residues results in a dramatic decrease or absolute loss of apoptosis-inducing ability or peroxidase activity of cyt-c. Other residues that are important in apoptosis and antioxidant activity of cyt-c are Lys7 and Arg38.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgments
We sincerely thank our reviewers for their critical reading and helpful suggestions. FA thanks the Department of Science and Technology (India) for financial support. SZ and MIH are thankful to the Indian Council of Medical Research for fellowship and grants, respectively. We apologize to those whose work was not cited or insufficiently cited because of space limitations.
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