Cytochrome C, Cardiolipin and Apoptosis

The purpose of this blog is to explain in brief the nature of the protein Cytochrome C and also to discuss it's place in the process of cell Apoptosis, or programmed cell death. I also hope to mention briefly a few pertinent details regarding the molecule Cardiolipin, it's structure and the interactions it forms with Cytochrome C during Apoptosis. In this entry the information is becoming more specialised. As of writing a general area of study has been decided upon for the research project. This area is to analyse the electochemistry of the compounds Cytochrome C, Cardiolipin and a specific cardiolipin/cytochrome C complex which forms during apoptosis. The exact queries have not been decided upon however. Rather it is hoped that through a review of available studies and data and preliminary voltammetric analysis of these compounds specific questions will arise which suggest a direction of study. Again the information is taken from a variety of sources. Primarily these are, "Molecular Biology of the Cell" 5th Edition, "Biochemistry" Garrett and Grisham and the scientific review "Free Radical Biology and Medicine", Volume 46, Issue 11, 1 June 2009 Pages 1439-1453. A typical undergraduate knowledge of Biochemistry is assumed.

To begin with a basic description of Cytochrome C, it's function and biochemistry is needed. Cytochrome C is a member of a general class of electron transport proteins. These proteins all contain Heme groups (the Fe containing group found also in Hemeoglobin). In humans cytochromes are restricted in location to the mitochondrial inner membrane where they form part of the electron transport chain of mitochondrial respiration. As such they are an extremely redox active series of proteins. As mentioned several classes of Cytochrome exist, including Cytochromes a, b, c, and e. The classification of the cytochrome is primarily dependent on the specific heme group attatched to the protein. Thus cytochrome C (CytC henceforth) posesses an attached c heme group. As mentioned cytochromes are primarily electron transport proteins. CytC specifically acts by moving electrons between Complex III and complex IV of the electron transport chain. An overview of the electron transport chain is given below to aid in visualising this position.

(1)







There are a small number of characteristics of CytC that should be mentioned. Firstly it is a relatively small protein, with a mass of 12,000 Da and consisting of the order of 100 amino acids. Secondly it is a relatively soluble protein, compared to the other repsiratory cytochromes. Thirdly, whilst it is found within the mitochondrial membrane it is not intrinsically bound to it. These factors will become important when cell apoptosis is discussed.







Cardiolipin is a phospholipid molecule found within the innner mitochondrial membrane. Specifically it is a double phospholipid which contains four fatty acid tails. The phosphate groups are bound to a central molecule of glycerol at the 1 and 3 carbon positions. In the membrane it forms a bicyclic structure such as is depicted below












(2)

The DAG segment represents the residue of 2 fatty acid chains. The fatty acid chains are typically about 18 carbons in length and unsaturated. As has been mentioned cardiolipin is found primarily in the inner mitochondrial membrane where it's main function is structural. By mass it can form up to 20% of the membrane itself, arranging itself into a bilayer. However it also has a small number of other primary functions. It acts as a structural component of several of the electron transport proteins, most prominently complex IV, where the presence of cardiolipin enhances enzymatic activity. Also because of the bicyclic structure of cardiolipin it is able to act as a proton trap controlling and buffering the pH of the intermembrane space of the mitochondria.

Thus far we have discussed the focus molecules in a basic form. The area of interest for the study I am undertaking relates to a specific interaction between cardiolipin and CytC that occurs during apoptosis. A full description of the factors and processes of cell apoptosis is beyond my means and desires at this time. I shall however describe one event that occurs, within a context. Cell apoptosis, or programmed cell death. There are many factors that can trigger cell death from within the cell itself, including DNA damage or oxygen deprivation. There also exist a series of regulatory proteins including mammalian Bc12 which regulate the start of apoptosis. The most important way this is done is by controlling the release of CytC from the cellular membrane. The release of CytC is the first step in a cascade reaction that results in the closing down of cell function and the activation of executionor proteins. Within this process though a specific reaction between CytC and Cardiolipin takes place.

After CytC has been released from the mitochondrial membrane Cardiolipin begins a migration into the cytosol and the following interaction takes place. CytC and cardiolipin will bond loosely. This triggers a conformational change in the CytC complex, revealing a new active site and allowing for enzymatic activity of the protein. Specifically the process transforms the complex into a peroxidase enzyme. The enzymatic process also somehow alters the structure and nature of the cardiolipin such that, when they dissociate, the cardiolipin cannot reenter the mitochondrial membrane. This in turn generates a porous nature in the membrane which contributes to cell death. The exact mechanisms of this process are amongst the details we hope to study in this research project. For instance questions such as "Does the cardiolipin dissociate immidiately following enzymatic activity, or does it stay bound?" are interesting ones I hope to ask. However until I can perform basic voltammetry on CytC, Cardiolipin and the peroxidase complex I cannot be sure what areas will be of greatest interest to me.

Notes:

1) Image Taken from http://en.wikipedia.org/wiki/File:ETC.PNG

2) Image taken from http://en.wikipedia.org/wiki/File:Cardiolipin_bicyclic_structure.jpg

Voltammetry

This Blog entry is intended to describe the basic processes of Voltammetry, and the application of this method to the analysis of enzymes. This information is summarised from the following sources: Elements of Molecular and Biomolecular Electrochemistry (Jean-Michel Saveant, pgs 298 to 316), Electrochemical Methods Fundamentals and Application (Bard and Faulkener, 2nd Edition). An understanding of chemistry equivalent to a typical undergraduate is assumed.

In analytical chemistry there are a wide array of analytical methods. These include mass spectrometry, spectroscpic methods, NMR analsis and so forth. In many cases such methods are innapropriate to the analysis being attempted. For instance if a compound is found in a solution that is optically dense i.e coloured spectroscopic analysis may not be useful. In this situation the solution would need to be seperated for the analysis to be performed. However if an analyte is redox active or is a an ion then it can be directly analysed using electrochemical methods. In particular voltammetry is useful in defining the characteristics of redox active molecules. This method is described below.

The basic instrument of electrochemical analysis is the electrochemical cell. A scematic of a simple cell is provided below.

(1)

Every electrochemical cell will contain 2 electrodes, a cathode and an anode. It will also contain an electrolyte solution, and a device to detect either current or voltage. In many cases, if the electrolyte for each electrode cannot mix the solutions will be connected by a salt bridge. In the case of electrochemical analysis one electrode will be designated the reference electrode and placed within a sealed area. This electrode is of a known type whose electrochemical characteristics are well established e.g. a hydrogen electrode. The other electrode is designated the sensor or working electrode and is placed in direct contact with the solution which is to be analysed. Voltammetry uses a standard electrochemical cell in it's processes. In addition this cell is connected to a power supply and an ampmeter. In this form of analysis a known potential (voltage) is applied to the sensor electrode and the resultant current is detected. This potential is then altered and the changes to current are recorded and analysed.

To describe what occurs let us suppose that the following reaction takes place.

O+e- --> R

That is the oxidised form of the redox active molecule being analysed is reduced at the electrode surface. Via mass transport the analyte moves towards the elctrode. When in contact with it the analyte is reduced and then moves away via mass transport. The current detected is the rate of electron tansport. This rate will depend on the temperature, the rate of diffusion of particles in the solution etc and can be represented mathematically by the Cottrell equation presented here. (2)

Where I=the current detected in Amperes.

n=number of electrons transferred

F=Faraday Constant, 96,500 C/mol

A=Electrode surface area, cm3

cjo=Initial concentration of analyte, j, in oxidised form, mol/cm3

Dj= Diffusion coefficient for analyte j, cm2/s

t= Time, s

The oxidation of analyte at the electrode, whereby electrons are transferred from the analyte into the elctrode is also possible. Which occurs will depend on the electrode potential. Should the potential applied be more negative than the reduction potential of the analyte it will be reduced. Should it be more positive than the reduction potential it will be oxidised. Should a negative potential be applied to the already reduced form then no current will be detected, the reverse being true about the oxidation of the reduced form. Finally the reduction of the analyte at the electrode will result in a negative current, the oxidation of the analyte will yield a posotive current. Whilst many methods of varying the potential exist the one most important to this study is cyclic voltammetry. In this method the voltage is varied in a linear fashion from more positive than the reduction potential to significantly more negative and back again. The resultant current is then recorded and analysed. The potentials are chosen to ensure both complete oxidation and reduction during the experiment to allow for a more detailed analysis. Typically the data can be presented graphically, such as below.

(3)

As you can see the potential is shown along the x-axis and the current along the y-axis. The shape shown here is distinctive of a single electron transfer. A wealth of information can be gained from such a voltammagram including the reduction potential, electron stoichiometry and so on. This will be demonstrated in a later post as a voltammogram is interpreted.

To conclude a brief mention shall be given to voltammetry as conducted upon enzymes. It is known that enzymes can achieve a direct electon transfer with the electrode surface without excessive difficulty. A more important factor is the problem of diffusion and mass transport for such massive molecules. Such problems can be overcom by adhering the enzyme to a specific membrane which is present at the electrode surface. The conditions for this are clearly explained in "insert source". In summary though, if a membrane is selected which is very thin (typically monolayer) and allows free access of the active site of the enzyme to both the solution and the electrode surface an electrochemical analysis can be achieved. What this means is that the structure within the enzyme appropriate to electron transfer (such as the heme group of a cytochrome protein) must be placed to allow a free flow of electons between the group and the electrode. Thus the membrane must not restrict this movement which can be achieved by direct access or conformational changes within the enzyme. A further advantage of this method (in addition to removing mass transport issues) is that a relatively small amount of enzyme is required for the analysis. This allows for meaningful analysis of enzymes which are available only in small quantities or at high prices.

Notes:

1) Image taken from http://www.doitpoms.ac.uk/tlplib/pourbaix/images/electrode_potential.gif

2) Equation image taken from http://en.wikipedia.org/wiki/Cottrell_equation. Checked against various sources.

3) Image taken from http://en.wikipedia.org/wiki/File:Cyclovoltammogram.jpg.

The major analytical focus of my research project is to study the electrochemical profile of various proteins, specifically enzymes. As such it may be necessary to give a brief overview of what an enzyme is and some basic structural details. This information is summarised from "Biochemistry (Zubay, 3rd Edition)" pgs 199 to 212 and "Molecular Biology (Robert F. Weaver, 4th Edition) pgs 32 to 38 in addition to any specific references in the body of the text. This is a general overview and will not cover the more detailed aspects of enzyme kinetics. Instead a more simple mention of the structure and pupose of enzymes will be presented. This assumes a background knowledge of Chemistry equivalent to a typical undergraduate student and is intended to provide background information for those who have not studied biology.

Enzymes are a specific subset of protein molecules. These molecules are large organic molecules composed of a chain of amino acid groups bonded by a peptide bond. There are 20 distinct naturally occuring amino acids, an adequate list of which can be found online via wikipedia(1). All thes amino acids posess a carboxyllic acid group (COOH) and an amine group (NH2) bonded to a central carbon atom. Each amino acid also posseses a distinct side chain bonded to the central C atom e.g. CH3 in alanine. It should also be noted that all amino acids posess a chiral centre at the central carbon, and thus exhibit optical isomerism(2). Despite this the naturally occuring amnio acids exist in the S-Isomer, except Cysteine which exists in the R Isomer(3). Finally, an amino acid can exist either in an unionised or ionised state. When ionised it forms a zwitterion.

A generalised strucutral image of both the ionised and unionised forms are shown below.

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As mentioned a protein itself is composed of a unique chain of covalently bonded amino acids. The exact length of this chain varies from protein to protein. Smaller ones may be composed of a few dozen amino acids, whilst larger proteins may contain thousands of individual amino acids. As the structure forms each amino acid is linked to the next via a peptide bond. Within the human cell this is usually an enzyme controlled reaction. A sample reaction to form a peptide bond is shown below.

(5)

Protein function depends greatly on the 3D shape of the molecule. A number of forces act on the molecule whilst it exists in it's straight chain form that drive it to form a specific structure. Several levels of organisation exist in protein structure, which will be summarised presently. The straight main chain is known as the primary structure.. After it is formed various interaction occur. A number of hydrogen bonds will form between nearby amino acid sub units of the protein, resulting usually in either a helix structure forming (known as an alpha helix) of a pleated sheet like structure (known a a Beta Sheet). In larger proteins different regions may exihibit one structure to another allowing both to exist. This level of organisation is called the secondary structure. The next level of organisation, the tertiary structure, is formed differently. This level of organisation exists due to the folding of the protein chain into a 3D shape. A number of forces exist whish contribute to this, and is largely dependent on the coposition of the side chains of the amino acids of the primary structure. The following 4 forces are some of the most important.

1) Hydrostatic forces. Various amino acid side chains present may be either hydrophobic or hydrophillic. Asumming an aqueous medium such as water this will cause the hydrophobic regions to be folded to the inside of the 3D shape and he hydrophillic regions to be found at the surface of the molecule.

2) Covalent bonding between amino acid side chains. It is possible for the constituents of the side chains to react with each other to form a covalent bond, forcing the molecule to fold to match it.

3) Ionic interaction/Dipole interaction. Many amino acid side chains exhibit either polar or fully ionic character. This results in repulsive and attractive forces altering the shape of the molecule.

4) Hydrogen Bonding. Further hydrogen between atoms brought into proximity due to protein folding will strengthen the strucure and cause further folding.

The Final level of organisation is known as the quaternary structure of a protein. This is where a number of seperate amino acid chains are bonded together to form a superstructure. Each sub unit is called a protein sub-unit. A schematic representation showng the connection between the structural levels is shown below.

(6)

Thus far I have spoken about protein structure in general. It is necessary though to refer specifically to enzymes. As mentioned before the structure of a protein is a major factor in it's function. Enzymes act as a biological catalyst allowing various reactions to take place more easily. Structurally this is possible due to the following. When the protein structure forms a specific region will exist possessing a distinct shape. This region is then able to interact with specific molecules in a certain way, often via hydrogen bonding. This interaction lowers the activation energy of a certain reaction which then can take place far more easily. In this respect they are very similar to inorganic catalysts. They differ from inorganic catalysts in the following ways.

1) They are highly specific. An enzyme will typically catalyse a very small number of reactions, usually of extremely similar molecules. By contrast inorganic catalysts such a platinum can often catalyse a vast range of reactions.

2) They are relatively unstable compared to inorganic catalysts. The function of an enzyme depends on it's 3D shape, specifically the shape of the active site of the enzyme. Small changes in the various forces that affect this, including temperature, pH and hydrostatic forces, can induce a change to this structure and deform the active site, limiting or even completely preventing enzyme activity.

3) Compared to simple inorganic catalysts enzymes are very large molecules. E.g Alcohol Dehydrogenase (an enzme that transforms alcohols into aldehydes or ketones) has a mass of 80 kDa.

There are many more aspects to enzyme activity, however this is hopefully a good basic introduction. In the next entry the basics of electrochemistry will be discussed, along with the application of these principles to the study of enzymes.

Notes.

1) The structural details were checked against "Biochemistry", Garrett and Grisham 3rd Edition and found to accurate. Link found at http://en.wikipedia.org/wiki/Proteinogenic_amino_acid

2) Excluding Glycine the simplest Amino acid, NH2CH2COOH. There is no chrial centre.

3) The side chain of cysteine contains an initial S atom, hence it exists in the R isomer.

4) Image Taken from http://en.wikipedia.org/wiki/File:Amino_acid_zwitterions.png.

5) Image taken from http://en.wikipedia.org/wiki/File:Amide_react.png

6) Image taken from http://en.wikipedia.org/wiki/File:Protein-structure.png

I am Peter Gearing, in my 3rd year as a student at the University of East Anglia student no 3169664.This Blog is being established for the purpose of recording and tracking the progress of my third year research project.

As of the current date (18/09/09) the complete details of the specific area of research being persued has not been decided on and thus only a more general outline of the project and the areas of study can be presented.In this entry I will outline some background information as the the type of research and the participants involved. The following entries will contain a more detailed summary of the scientific background relating to this project. After this as the specific area of research is decided the methodology will be entered, as well as significant results obtained during the study and from experimentation.

The supervisor for this project is Dr Julea Butt from the University of East Anglia. A title of the area of interest is "The use of Cyclic Voltammetry to study Immobolised Enzymes." What this means is that, using various techniques it is possible to take an enzyme filled solution and immobolise a certain fraction onto a surface. This surface containing the enzyme can then be subjected to any number of analytical techniques. However the one used in this study is Cyclic Voltammetry. This means that a potential difference (or voltage) is applied to the enzymes. This Voltage is then altered in a steady or cyclic fashion. The enyme will then be either oxidised or reduced, dependant on the Voltage applied. By measuring the resulting current a great deal of information regarding the enzymes can be recorded.

The next Blog entry will be a short mention of the biological function of Proteins. Following this will be an entry discussing the use of Cyclic voltammetry and the advantages of immobolised proteins. This should cover the background information necessary to understand this project and the area of interest.