LPandiscia+Final

**Cytochrome c binding to liposomal surfaces**
Leah Pandiscia Drexel University Department of Chemistry Drexel University, Philadelphia, PA 19104 Submitted December 1, 2011

Cytochrome //c// is a small heme protein located in the inner mitochondrial membrane (IMM). It is involved in both the electron transfer process and apoptosis. Ferricytochrome //c// in solution can adopt a multitude of conformational states; from the fully folded native state through the partially unfolded to a fully unfolded state. The majority of mitochondrial cytochrome //c// interacts with anionic phospholipids on the surface of the inner membrane. Liposomes are used as a model system to mimic these anionic phospholipids. The binding of cytochrome //c// to these phospholipids involves three different sites on the protein’s surface, two of which bind electrostatically, while the other one binds through hydrogen bonds. The modes and constants of protein-membrane binding constants to these sites depend on the molar ratio of the protein and membrane binding sites, pH, ionic strength, and liposome composition. The conformational characteristics of cytochrome //c// binding to membrane surfaces dictates the role that cytochrome c plays in the mitochondria, either as an electron carrier in the electron transport process or as an apoptotic initiator. As the binding of cytochrome //c// to cardiolipin (a common mitochondrial phospholipid) destabilize the protein/membrane complex, the crevice within the protein allows for hydrogen peroxide to access the heme pocket and lead to peroxidation. The peroxidase activity facilitates the release of cytochrome //c// from the membrane surface and its release into the outer mitochondrial membrane, becoming active in the apoptotic pathway.

Cytochrome //c// is a relatively small heme protein (see Figure 1) located in the inner mitochondrial membrane (IMM).Cytochrome //c// carries electrons between cytochrome reductase (complex III) and cytochrome //c// oxidase (Complex IV) in the electron transport process via aerobic respiration[1]. The interest in this heme protein has recently been renewed through its association in triggering apoptosis. To prompt this event, cytochrome must first detach from cardiolipin lipids of the inner membrane. After apoptosis regulator proteins permeabilize the outer mitochondrial membrane, is released into the cytosol. Here, cytochrome c interacts with apoptotic protease activating factor-1 (Apaf-1) which activates caspases and later the disassembling of the cell [2].
 * Introduction**

The release of cytochrome c requires a decrease in the degree of interaction between cardiolipin (CL) fractions of the inner membrane and cytochrome //c// in order for the protein to detach from the membrane surface. Therefore, the mechanisms by which cytochrome //c// and CL interact determine whether the protein will facilitate the electron transfer process, or dissociate from the membrane surface and be released into the cytosol to trigger apoptosis [2].

In order to understand this initiation process of apoptosis, the conformations that cytochrome //c// assumes on liposomes must be thoroughly characterized. The disruption of the protein-membrane binding must then be explored, since this process is necessary for the oxidation of cardiolipin. This paper will focus on the thermodynamic states of cytochrome //c// in solution, which provide a comprehensive model that can be applied to cytochrome //c// on membrane surfaces, and the modes of protein binding to membrane surfaces. These characterizations will describe what initial conformational state the protein must adopt for it to dissociate from the membrane surface.



In order to understand the behavior of cytochrome //c// when bound to membrane surfaces, the multitude of conformations cytochrome can adopt in solution must first be determined, characterized and understood, a project currently under investigation by our group. This characterization of cytochrome //c// min solution will provide the framework for the interpretation of the data obtained on liposome bound cytochrome //c//. Conveniently, the covalently bound heme group allows different conformational states of the protein to be probed by a number of spectroscopic techniques.
 * Thermodynamic states of cytochrome //c//**

Theorell and Åkesson were the first to describe the five classically distinct protonation states of ferricytochrome //c//, whose occupations are dependent on the pH of the solution. In ferricytochrome //c//, state I is an unfolded, denatured state, which is populated at acidic pH (>2). The protein was later found to be a statistical coil at low ion concentration and a molten globule at high ion concentrations. In state II, the protein is partially unfolded at pH 2-3 and has a mixture of two substates: one whose heme group is penta-coordinated [4] and the other whose heme is hexa-coordinated and bound to histidine 33 (His33) [3]. In state III the protein’s heme iron is low spin, hexa-coordinated state owing to the occupation of the sixth coordination place by the sulfur of the Met80. This state is considered as the fully folded, native state, which is predominantly populated at pH values between 4.5 and 8.5 [4]. The transition from state III to IV, the Met80 is replaced by a lysine residue (Lys79 or Lys73) and is more flexible than the native state. With the exception of the molten globule state (1), in all other states the heme remains coordinated to proximal histidine 18 (His18). State V occurs under more alkaline conditions and is again considered to be partially unfolded and hexa-coordinated to leucine 68 or a water molecule, depending on solution conditions [4].



A misligated protein state (termed B2HH) was later discovered. In this state, a histidine ligand replaces the methionine ligand bound to the heme iron. B2HH is a partially unfolded via denaturing conditions, such as 4 to 6M guanidine chloride, at neutral pH. In the presence of this denaturant, the Met80 ligand is replaced by His33 and the complex forms a hexa-coordinated low spin state. This unfolded state is accompanied by a complete loss of the secondary and tertiary structure of the protein [6]. If the protein is refolded at neutral pH, the partially folded state with two histidine iron ligands is temporarily populated as kinetic intermediate. It suggests that the misfolded states with two histidine ligands have to convert to penta-coordinated states prior to the folding process [7, 8].

Recently, an extension of the classical Theorell and Åkesson model has been suggested based on the discovery of a thermodynamic intermediate state, which becomes populated at mildly alkaline conditions (pH 9) and low anion concentration. In this intermediate termed III*, the Fe-Met80 bond is weakened but still remains ligated [9]. There is also another thermodynamic intermediate at acidic pHs (pH < 6). Here the heme is hexa-coordinated and ligated to His18 and water [10].

While the conformation of ferricytochrome c is pH and temperature dependent between pH 1 and 12 and temperatures between 273 K and 373 K, ferrocytochrome c is locked into the same native conformational state between pH 3 and 12 and temperatures below 370 K. Ferrocytochrome c also shows much greater thermal stability than the ferri form as well [11]. In ferricytochrome //c//, as temperature increases, the Met80-iron III bond of the heme becomes increasingly disordered. At high temperatures, ferricytochrome //c// is in a hexa-coordinated low spin state where the His18 ligand remains intact. The heme pocket of the protein is now open but there are only slight changes to the secondary structure [12].

Rytomaa and Kinnunen discovered that cytochrome c binding can occur at different sites on the protein, termed A (anionic) and C (cardiolipin). Site A is located next to lysine 72 and 73 of cytochrome //c// (see Figure 3) [13]. These two binding sites were initially discovered when the authors found that binding affinities for the membrane surface depend on pH. At pH 7.0, cytochrome //c// is displaced from cardiolipin-containing liposomes via the competition between nucleotides and cardiolipin for a binding site on the protein. At pH 4.0, however, salts and nucleotides do not dissociate cytochrome //c// from the liposomal surface thus indicating that the interaction between protein and liposomal surface takes place via hydrophobic binding [14]. The interactions at site A are electrostatic and therefore occur via peripheral binding, or the temporary interaction of a protein with the periphery of a membrane [13, 14, 15]. The binding of site A is dependent on ionic strength and involves the binding of deprotonated acidic phospholipids to positively charged (lysine) patches on the surface of the protein. A second site, site C, is located next to asparagine 52 on the protein and binds protonated acidic phospholipids through hydrogen bonding [14]. A third binding site on cytochrome //c// was recently obtained. This electrostatic binding location, named site L, consists of lysine residues 27 and 22 and is adjacent to site A on the protein (see Figure 4) [16].
 * Cytochrome c binding to membrane surfaces**





Based on the discovery of these three different binding sites, the existence of two pools of cytochrome c has been suggested. The first of cytochrome //c// is loosely bound and sensitive to electrostatic interactions, pH, ionic strength, and surface charge density. The second pool consists of proteins tightly bound to the inner mitochondrial membrane and interacts with complexes involved in the electron transfer process. A mechanism for extended lipid anchorage involving this second pool of cytochrome //c// was also presented by Wallace and coworkers [18]. A cardiolipin acyl chain bound to site C of the cytochrome //c// must first make its way through the protein’s polar region and slip into a channel aligned with hydrophobic amino acid residues in order for the acyl chain to insert itself along the heme crevice. The other acyl chain remains hydrophobically anchored to the membrane surface [19]. This insertion of an acyl chain into the interior of cytochrome //c// induces conformational changes of the protein, which involve the loosening of the protein’s tertiary structure [18].

The mode of membrane binding to cytochrome //c// depends on the liposome/protein molar ratio. Oellerich et al. varied the ratio of lipid to protein concentrations to describe these modes. At high lipid to protein ratios, peripheral binding by electrostatic interactions dominates and the protein is in the B2 conformation substate. In B2 substates, the Met80 is replaced by water as the sixth heme ligand [15]. The degree of protonation of acidic phospholipids increases with increasing surface charge density or the molar amount of liposome, and the mode of membrane binding favors site C [13]. With increasing protein surface coverage, the electrostatic interactions weaken and a low spin state is preferred. The protein now has the ability to integrate into the membrane surface itself and bind with the hydrophobic core. This method of binding is called integral binding, or membrane anchoring, and now competes with peripheral binding.

At high protein concentration, the liposomal surface can be fully covered by a protein monolayer. Beyond full coverage of the liposome surface, additional binding of cytochrome //c// to the protein monolayer forms a second protein layer, which then affects the shape of the protein-bound liposome surfaces. Any further protein binding causing phase separation, flocculation, and sedimentation of the liposome from aqueous buffer solution. All three of modes of binding explored by the authors induce some degree of protein unfolding to acquire their final states [15].

Cytochrome c must first begin its detachment from cardiolipin and the IMM surface by unfolding before it can be released into the cytosol. Pinheiro et al. showed that the unfolding of cytochrome c at a membrane surface follows a pathway involving a native-like intermediate with Met80 replaced by an alternate ligand. Initially, the contact of native cytochrome //c// and lipid membranes is driven by electrostatic interactions between the positively charged amino acid residues of the protein and the negatively charged headgroups of the lipid on the membrane surface. The high density of the headgroups on the membrane surface creates an electrostatic potential, which attracts protons, thus decreasing the local pH at the membrane surface and inducing protein folding. The authors discovered that the native conformational state of cytochrome //c// transitions into a more denatured-like state, which retains the native-like secondary structure but lacks the tight packing of the tertiary structure and ligation of the Met80 ligand. Therefore, the protein must proceed through this misfolded state where Met80 becomes dissociated from the heme before the subsequent unfolding events can occur [20].

The idea that cytochrome //c// is present in the mitochondria as loosely and tightly bound populations attached to the inner mitochondrial membrane via cardiolipin supported the following proposal by Ott and coworkers. They stated that the cytochrome //c// release from the mitochondria requires a two step process, and depending on the conditions within the IMM, both populations of cytochrome c can detach from the membrane surface. If there are changes in the pH, ionic strength, or surface charge density, the loosely bound pool of cytochrome //c// can be mobilized. If cardiolipin is oxidized in the IMM, the tightly bound population of cytochrome //c// can be mobilized. Cytochrome //c// must first detach from the mitochondria membrane by breaking its electrostatic and hydrophobic bonds with cardiolipin, thus rendering the protein soluble. By increasing the ionic strength, the degree to which the electrostatic interactions are disrupted determines the amount of loosely membrane-bound cytochrome //c// released. The changes in the membrane structure via the peroxidation of cardiolipin determine the amount of tightly bound cytochrome c released. Once the protein has dissociated from the inner membrane, which requires the peroxidation of CL, the permeabilization of the outer mitochondrial membrane allows the protein to pass into the cytosol [21].

Recently, the Kagan group illustrated that before cytochrome c binds to Apaf-1 to initiate apoptosis, it gains peroxidase activity while remaining bound to the inner mitochondrial membrane surface. The authors supported the work of Ott et al. and confirmed that cytochrome //c// acts as a catalyst of cardiolipin oxidation, which is required for both cytochrome //c// detachment from the inner mitochondrial membrane and for the permeabilization of the outer membrane [22]. In order for cytochrome //c// to achieve peroxidase activity, the iron of the heme must remove oxygen from hydrogen peroxide to form a ferryl complex. The heme crevice of the protein must be disrupted enough to allow the hydrogen peroxide access to the iron [22]. Diederix et al. discovered that under low denaturant conditions, the peroxidase activity of cytochrome c in solution is increased. The authors also noted that the strength of the axial ligand replacing the Met80 ligand has an effect on the extent of peroxidase activity [23].

The peroxidase activity of cytochrome c binding to cardiolipin was investigated by Belikova et al. The peroxidase activity of cardiolipin/cytochrome c complexes is maximized when there is an increase in the accessibility of the iron atom in the heme crevice of the protein [24]. These findings indicate that the protein undergoes a transition into a non native conformational state to allow the protein to become a peroxidase [22]. In order for hydrogen peroxide to interact with the iron, the iron-Met80 bond must either be disrupted or broken, where the latter option generates more peroxidase activity. The electrostatic interactions between cytochrome //c// and cardiolipin initiate the peroxidase activity of cytochrome c when electrostatically bound to the inner mitochondrial membrane. The hydrophobic interactions are needed to stabilize the unfolded conformational state of cytochrome on the cardiolipin membrane surface [25].

The interaction of cytochrome //c// with membrane surfaces in the mitochondria has yet to systematically be characterized. The research conducted thus far has revealed that it is not the native state of cytochrome //c// that is involved in peroxidase activities. On anionic surfaces, the misligated B2 statesand its derivatives are the functional states of the protein for its role in peroxidase activity and thus apoptosis.
 * Conclusion**

This study will provide the characterization of the conformational changes in which the protein adopts on the surface of anionic phospholipid containing membranes with the presence of cardiolipin, thus providing a complete picture of the structure and thermodynamics of cytochrome //c// on the surface of phospholipid vesicles. The study will involve varying the lipid composition and concentration, pH, ionic strength, and temperature. This study will also demonstrate a qualitative set of information to illustrate its conformation in cytochrome //c//-dependent apoptosis, namely the oxidation of cardiolipin in the presence of hydrogen peroxide. This complete characterization can ultimately be used to help understand its physical role in biological processes and disease states.


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