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 * Understanding Cytochrome c* ** ** (*relatively)

** Jonathan Soffer Drexel University Department of Chemistry Drexel University, Philadelphia, PA 19104-2876 //Submitted//: December 5, 2009 (Chemical Info Retrieval class)

I. I NTRODUCTION II. C YTOCHROME C (IN SOLUTION) III. C YTOCHROME C (ON MEMBRANE SURFACE) IIII. S UMMARY AND OUTLOOK **
 * C ONTENTS

For well over the last six decades biologists, chemists and physicists alike have toiled with the fundamental question of how cytochrome c works. Cytochrome c is a small soluble heme protein (MW ~12.4 kDa) that is associated with the mitochondrial inner membrane space (1). It is the highly essential mediator of the electron transport chain from cytochrome reductase b-c1 complex (complex III) to the cytochrome c oxidase a-a3 complex (complex IV) (2). It is through this oxidative pathway of the electron transport system that this protein plays a significant role in the production of adenosine triphosphate, ATP, a high-energy intermediate found and used by all living cells. Cytochrome c has played another role serving as a model protein for the study of electron transport (2) as well as provided great insight with the process of understanding protein folding (3-6). Theorell et al originally characterized the conventional understanding in the conformational changes of cytochrome c in 1941 (1, 2, 7-11), in which the five states (I-V) of the protein were thoroughly described. The intermediates of these five states have been heavily debated over the last few decades as new techniques emerge in the study of this protein. Recently, the importance of cytochrome c has reemerged due to its association in the aggregation of α-synuclein, which is responsible for Parkinson’s disease (12) as well as the pivotal role it plays in the apoptotic process (13, 14), which is programmed cell death. Even with this vast amount of information that has been collected there is still a great deal that is not thoroughly understood, in particular when looking further into its biological mechanism, as well as the nonnative conformational state of the protein that participates in this important biological process.

Cytochrome c has been extensively used as a model system for protein folding studies. As with most proteins its structure lies primarily in the carbon backbone, the heme and most importantly with the ligands that are associated with the heme. The protein heme interaction dictates the functionality of the protein but this is also useful in that it serves as a spectroscopic marker (15, 16) when studying the conformational changes that protein undergoes under a variety of conditions, such as pH or temperature changes.
 * I ** ** NTRODUCTION **

The peptide chain is composed of 104 amino acid residues and is attached to the heme by means of a covalent thioether linkage [R-S-R’] between two cysteines, Cys-14 and Cys-17, and axial ligation of the heme iron is coordinated to a histidine and methione, His-18 and Met-80, in the native state, state III at pH7 (1). The secondary structure and heme environment of the functional heme group in the native state is shown in //figure 1.1,// with horse heart cytochrome c presented as determined by Bushnell //et al//. The secondary structure consists primarily of helical units, shown in purple in //figure 1.1//. These helical units are broken down further into three major and two minor helical structures. These structures are then further connected by a polypeptide linkage and folded into a pocket where the heme prosthetic group is located. Other then these major structures a significant part of the secondary structure relies on the other small amounts of common secondary interactions, such as hydrogen bonding, of which there are only three in the main chain, as well as a very small two stranded anti-parallel β -sheet interaction, shown in yellow, found in residues 37, 40, 57 and 59 (1).


 * F IGURE 1.1 : ** Horse heart cytochrome c, as reported by Bushnell et al. (PDB: 1HRC) (1), this figure was produced using VMD software (17). With the central heme iron illustrated with it van der Waals radius presented.

Investigating into the linkage of the heme prosthetic group, protoporphyrin IX, there are only four amino acid residues holding the prosthetic group in place, as just mentioned. This prosthetic group is just exposed to the outside solvents or in the mitochondrial inter-membrane space by only four atoms. These amino acid sequences are situated holding the prosthetic group on the front edge (1), as shown in figure 1.2, so that the four atoms are exposed. The amino acids that are exposed on the edge of the protein are the positively charged arginine and lysine residues. It is believe that these groups are responsible for the formation of complex interactions with their associated redox partners into the proper orientation of the heme group prior to an electron transfer event, with lysine being the residue that is actually binding. Only a mere 7.5% of the total heme surface is exposed to interact with the surrounding solvent or mitochondrial inter-membrane space (18). The protein heme interaction and solvent exposure dictates the functionality of the protein but this is also useful in that it serves as a spectroscopic marker (15, 16) when studying the conformational changes that protein undergoes under a variety of conditions, which will be briefly evaluated in further detail. 
 * F IGURE 1.2 : ** Horse heart cytochrome c, as reported by Bushnell et al. (PDB: 1HRC) (1), this figure was produced using VMD software (17). With the central heme iron illustrated with its van der Waals radius presented and each residue (14, 17, 18, 80) of attachment shown in Corey, Pauling and Koltun (CPK) mechanical modeling form.

The physical interaction domains located on the surface of cytochrome c for its redox partners have been shown to involve positively charged lysine residues around the exposed heme edge. The non-native states of cytochrome c were determined to play an important biological role (13, 21), leading to a continuous interest in the transition from the native state (State III, pH 7 at room temp) to alkaline and acidic states, I-V (8), in which the Met-80 ligand of the heme is replaced (22), this is illustrated in //figure 1.3//. However the details of this landscape are still not fully understood. A consequence of this movement, in particular is seen with its redox partners allowing for a change in functionality and further minimizing any rotation of the protein. 
 * F IGURE 1.3 : ** Proposed energy landscape of ferricytochrome c across the pH range and increase in temperature (23). Question marks denote proposed or unsure ligation to the heme iron.

The unfolding or non-native conformations of the protein can be established through a variety of techniques, using denaturants (eg. urea, peroxide), through alteration of the pH, or through temperature changes. Thermal unfolding depends on the oxidation state of the heme iron, with the ferri state, Fe(III), being much less stable then the ferro state, Fe(II). Investigations into the unfolding process generally focus on the ferri state. This is generally described as a two-step process with intermediates populated above 333K (24). In alkaline transitions this involves the replacement of methionine 80 with one of the lysine residues in the vicinity, in the alkaline transitions this translated into ligation of Lys-73 or Lys-79, and the formation of a more open heme crevice (25). The acidic transitions are less understood, as the alkaline transitions were more heavily studied, but for these traditions all that is known experimentally is that the methionine linkage is broken (23). This is something that is not fully understood and under current investigation. It has proven to be more difficult to identify experimentally either the structure of the site of interaction or the actual mechanism of electron transport in these protein complexes. Much of what has been experimentally determined about the functions of these molecules has been from studies comparing structural, physical and biochemical properties (26). With experimentation focusing on the mechanism of the electron transport chain, associated disease states providing much insight into the biological interaction of cytochrome c and the other associated membrane bound carrier proteins. The precise chemical nature of the transfer of electrons through this system as well as the agreement of what is actually occurring is still highly controversial.

The majority of the studies conducted thus far have investigated the protein in solution. These investigations paid little attention to the solution environment, generally placing cytochrome c in a high ionic strength environment. This was performed because conformational changes can be induced through denaturants such as urea, pH changes, or temperature changes. This was due to the fact that the investigators were studying the induced conformational changes occurring, and less into the biological nature of these structural events. Current studies have moved onto the conformational changes of cytochrome c under low ionic strength taking into account more biologically relevant related conditions.
 * C ** ** YTOCHROME C (IN SOLUTION) **

This lengthy past has provided much insight into a variety of spectroscopic techniques that have been used and are being used to gain a deeper understanding into the protein, and with this the modifications that are being made to the protein can be made fast are reversibly. Each of the spectroscopic methods employed provides further information and deeper understanding into the structural changes that are associated with the heme group that could furthermore provide information on the possible structure changes that are occurring at the redox reactions sites (27). In addition to the crystallographic methods, UV-Visible Spectroscopy, Fluorescence, Circular Dichroism (UV/VisCD, VCD, SRCD), Raman, NMR, EPR, Stopped-flow, and molecular dynamics have all been employed to further add to the extensive library of information the cytochrome c provides. These techniques will be briefly reviewed, as they have been significant in the current understanding and for the further investigations when dealing with cytochrome c as associated with the surface of a membrane.

//UV/Visible spectroscopy// Through the use of UV/Visible spectrophotometry it has been determined that when in its oxidized state cytochrome c has a band that arises in the red region of the spectra at around 655 mμ and continues to a maxima of 695 mμ. Theorell and Akesson in 1939 were the first to investigate this band in great detail and this still acts as an identifying marker into the oxidation state of the heme iron (7, 8). The disappearance of this band was also illustrated at a pH lower than 2.5 and at greater than 9.35 (28). The absorption spectra of cytochrome c illustrate the percent relative light absorption of both it’s oxidized and it’s reduced state. The disappearance of this band was later determined to occur when the complex of ferricytochrome is made with the cyannide of azide complexes of associated amino acids found within the protein. Further experimentation of this band provided information into the sensitivity of the band with variation to temperature and its absence when present in a polymeric form (28). It was later determined that structural parameters other than the chemical nature of the heme iron ligands must be involved with the transitions that are responsible for this 695 mμ band. This 695 band, part of the charge transfer band, became of particular importance for use as a marker in resonance raman spectroscopy. In the spectrum of ferricytochrome c the charge transfer (CT) band located at 695 nm is highly asymmetric (29). This indicates conformational heterogeneity due to the coexistence of different conformational sub-states. Later interpretation being association of the observed sub-bands of the charge transfer band being assignable to different dπ, Fe(III) transitions.

//Resonance Raman spectroscopy// Through Resonance Raman spectroscopy further structural and functional roles of cytochrome c was developed where the other methods failed to explain anything beyond the overall function of the molecule and not related to the intrinsic nature of cytochrome c itself. Through this method the vibrational spectra can be selectively probed to provide some more direct information into the possible structure changes that arise during the oxidation-reduction reactions at the heme prosthetic group. As Thorell and Akesson illustrated ferricytochromes c to exhibit five discrete pH-dependent conformational states (7, 8), with the alkaline conformation formed through the raising of the pH to an alkaline values (pKa = 8.5-9) have recieved particular attention with various instrumental analysis as well. The interest in this form on particular of the cytochrome c protein has brought forward further evidence that cytochrome c undergoes the conformational changes upon the binding to other proteins in the manner previously described, the two state form that has yet to be proved. The alkaline isomer of this protein also posses a six-coordinate low spin heme iron that furthermore maintains the native imidazole asparagine, N, ligand associated with the histidine 18, His18, while the sulfur atom of the methionine 80, Met80, also previously described becomes replaced by a strong field. With the alteration of pH within the range of 7 to 10 the dichroic pattern in the intrinsic absorption region is altered to a smaller extent. But the axial ligands that are present in the heme iron are bound in the oxidized form of cytocrome c and allows this residue to be susceptible to a substitution by an endogenous ligand at a high pH or at a high temperature forming an alkaline isomer (30). In this isomer form the iron still maintains its low spin (Fe III) state but is accompanied with a spectroscopic change as well that presents itself as a loss of the 695 nm band as well as a shift in the resonance spectra, H-NMR and Raman which show an alteration in the vibrational modes of the heme pocket. The wealth of data that has been quickly described above brings the conclusion that this change in the heme pocket is that of the methionine 80, Met80, with a surface lysine in axial heme ligation (30, 31). This was the source of the dispute that had been seen across the various disciplines for well over forty years, and has finally been somewhat resolve to be due to a nearby lysine residue, as dictated in figure 1.3.

//Circular Dichroism spectroscopy// An irreplaceable tool in conjunction with absorption spectroscopy has been circular dichroism spectroscopy [CD], which has been useful in the rapid characterization and identification of different conformational states of cytochrome c. The information from this method has provided insight into the intactness of the heme environment, which is now the standard method of detection, especially with relation to cytochrome c. For this visible CD measurements focus on the B-band region, between 22,000 and 26,000 cm -1 mainly due to its large intensity (32). With relation to the oxidized and the reduced species there is a dramatic difference with the CD spectra. In looking at the oxidized state there is a very pronounced couplet that occurs at neutral pH. It was surprising to me that there were limited investigations into the understanding of this from a theoretical approach. However, as the environment is altered into a nonnative state this couplet is broken being resolved into a positive Cotton band.

This final method helps to bridge the gap in the determination of the mechanism of cytochrome c was through the use of circular dichroism spectroscopy for the determination of biomolecules. Since nearly all molecules synthesized by biological organisms are optically active. This method proved to be very useful in the determination of a larger number of proteins through the use of a quick low resolution technique for the determination of structure, through this a protein with a known structure can be tested in order to determine a basis set for secondary structure merely with the alteration of pH and temperature. Circular Dichroism spectroscopy can resolve individual transitions as separate bands, thus the spectra obtained are immune from interference by free metal ions, as only the protein bound species are observed and not those of other outside forces (33), this is a remarkably useful tool.

Overall these spectroscopic studies have provided significant evidence that cytochrome c undergoes a conformational change upon its binding to cytochrome c oxidase. These tools provide a useful understanding of the conformational changes that the protein takes under various conditions; the next step is the start of how this protein then interacts with the surface of the membrane. The methods just described, that were well established over the years, is currently being used to gain a better understanding of what is occurring on the membrane surface. As well as to get a better idea into the physiological changes that this protein undergoes.

The native state of cytochrome c occurs at physiological pH with the central heme coordinated to the histidine 18 and methionine 80 as previously mentioned, and illustrated in figure 1.4, with the orientation that it takes when bound to the surface of a membrane through positive lysine patches on its surface to the negative patches of the membrane. Interaction of cytochrome c with the negatively charged lipid membranes, such as CL, induces a considerable disruption of the native compact structure of the protein and induces intermediate conformations between the native and the unfolded states (34) It is increasingly unclear at this point in time whether the native state is the one of physiological relevance or not. However it is becoming more apparent that cytochrome c does change its structure upon binding to negatively charged lipid membranes, with cardiolipin, CL, being the most effective binding vector (34-36), initial studies, unpublushed at this point lead me to believe that it takes on state IV as a positive cotton band is seen in the CD measurements.
 * C ** ** YTOCHROME C (ON MEMBRANE SURFACE) **

** F IGURE 1.4 : ** Horse heart cytochrome c, as reported by Bushnell et al. (PDB: 1HRC) (1), this figure was produced using VMD software (17). Illustrated as though bound through the lysines on the proteins surface.

This cardiolipin interaction with cytochrome c is of particular interest as it has been found to form a peroxidase complex that catalyzes CL oxidation during apoptosis (34). This process is pivotal in the mitochondrial stage of the carrying out of the programmed cell death. The electrostatic interactions that have been seen between cytochrome c and the lipid membrane is not the only factors that affect the unfolding of the protein. Interactions of proteins and lipids are believed to be the key factor in the determination of the structural and functional characteristics of membrane proteins. // Fl //// uorescence Resonance Energy Transfer [FRET] // Studies of cyt c binding through the use of FRET using labeled lipids to heme have confirmed the presence of two modes of interaction, the first of which is an electrostatic low-affinity binding to deprotonated CL molecules and a high-affinity binding stabilized by electrostatic and H bonding to a partially protonated CL (37). This is believed to have a direct effect into the controlling of biological redox reactions via changes of the transmembrane potential (38).

Resonance Raman again found Met80 bond disruption upon binding that was further advanced by analysis of the heme configuration revealing the coexistence of a mixture of hexa-coordinated low-spin iron and high-spin iron further coordinated to the protein lipid interaction (34). Due to high-affinity binding of CL with cytochrome c it is understood to be due through an electrostatic interface between negatively charged phosphates on CL and positively charged lysines on cyt c, as well as through hydrophobic interactions of CL's acyl groups with a hydrophobic domain of the protein (34). This process again was verified through CD spectroscopy.

These high affinity interactions between cytochrome c and phospholipids (anionic) are now thought to involve the binding at two different sites to the membrane, one of which is the partially unfolded state as just mentioned. The other is through a partial insertion of the protein into the membrane through interation with CL (39). The structural transitions of cytochrome c found the opening of the heme crevice, as detected by CD and disruption of the Met80 residue with the heme iron, detected by UV/Vis and Raman spectroscopy. There also is the emergence of a penta-coordinated high-spin heme that is not known in solution that was presumed to be related to histidine 33 (34), but further studies must be conducted to verify this. It would be interesting to see which state this correlates to in solution or if it the emergence of a new intermediate related solely to the protein lipid interaction.

The native state of cytochrome c, state III, favors its most common function as an electron shuttle between complexes III and IV of mitochondria. However, new evidence provides an additional argument that cytochrome can form two types of complexes with a membrane, and cardiolipin in particular. This is where either the partially protonated or deprotonated cardiolipin being responsible for the proteins surface adsorption, relying on electrostatic interaction. Although not yet known one of these leads down the pathway of peroxidase activity and apoptosis. Experimental estimation of the heme-bilayer distance suggests that at physiological pH 7, that cytochrome c is located in the lipid-water interface in both types of complexes, while at pH below 6.0, the energy barrier for the protein insertion into membrane core is possible (39).
 * S ** ** UMMARY AND OUTLOOK **

It is even more interesting that despite all of the structural information that is known about cytochrome c, as well as the basis for the perturbations that a fundamental controversy still remains regarding the nature and role of observed oxidation state dependent conformational changes as well as with their relationship with the electron transfer event (10).

We are in the process of witnessing the collapse of an old dogma of biochemistry. The linearly thought one gene is used to produce one protein which controls one function. As new functions of cytochrome c are determined we have fully seen this paradigm shift in action.


 * A ** ** CKNOWLEDGEMENTS **

In figures 1.1-1.2 the secondary structure was determined using the VMD software package (17) applying the STRIDE algorithm (19, 20) which uses a heuristic algorithm to determine these secondary structure types through a combination of hydrogen bond energy and tortional angle information, which is statistically derived. This is then optimized for agreement with the crystallographic designations. This crystallographic information was obtained using the protein database, (PDB ID: 1HRC) information was based primarily on protein crystallographic information, via x-ray structural analysis (1).

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