Protein structure plugins for OSX

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Almost a year ago I made a post about some quicklook plugins that I was working on. At the point in writing, I essentially had a finished product with the exception of a bug that I was unable to track down. I became busy with my studies and other projects and in effect left the project alone for some time. A few months back I had some spare time to pick up the project again and ended up rewriting the plugins from scratch with OSX 10.7 in mind. In doing so, I’ve created two quicklook plugins for OSX 10.7. One plugin is for PDB files and at the moment uses pymol on the command line in /usr/bin or /usr/local/bin (a symlink will also work) as the backend renderer and caches the images to /tmp, which are deleted on reboot. This plugin is somewhat sluggish and can take up to 30 seconds to render an image; this is due to pymol only allowing raytraced rendering on the command line. In order to get around this, I would like to, in the future, code up a small lightweight molecular renderer that can parse files such as pdb and chm and provide a number of command line rendering option.

 

The other plugin I wrote up is for the purpose of displaying x-ray diffraction data without having to open up external applications. The plugin falls back on the DiffractionImage package in CCP4, in which I pulled out the code responsible of generating jpg images, hacked up the command line arguments a little, compiled and added it as a resource to the plugin.

 

The software:
Current version: 0.1
Compiled version can be downloaded here
Source code can be downloaded here

 

Install instructions:

1. Copy an paste the osc and pdb.qlgenerator files to ~/Library/QuickLook/
(If the folder does not exist, create it)

 

2. In order to get the pdb plugin to work, you need to create a symbolic link to pymol
 For this you will need an x11 version of pymol, this can either be the macports/fink or the MacPyMOL.app version.

 

Macports:
 sudo port install pymol
 Find the location, with "which pymol" - Probably somewhere like /opt/local/bin/pymol
 ln -s <pymol location> /usr/bin/pymol
Application:
 Others might be using a version of pymol in the applications folder.
 This by default is not an x11 version, but by renaming it to PyMOLX11Hybrid.app, it will enable X11 mode.
 cp -R /Applications/MacPyMOL.app PyMOLX11Hybrid.app
Create the symbolic link
 ln -s /Applications/PyMOLX11Hybrid.app/Contents/MacOS/MacPyMOL /usr/bin/pymol

 

3. If the plugins do not work immediately, you can refresh the quicklook daemon with:
 qlmanage -r

 

Screenshots:

Using OSX features for structural biology

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While delving into the art of structural biology I found that I had started to collect pdb and x-ray diffraction files. After a while, keeping track of pdb files starts to become quite tedious and results in one having to open up pymol or another application of choosing to see what the protein is.

To remedy this situation, I decided to write up some plugins for osx that make use of spotlight and quicklook. The spotlight plugin is capable of pulling out the metadata from the pdb file and store it in an xml database, which allows for the instant identification of a pdb file without needing to open the file. I also thought it would be nice if you could visualise the protein structure, so I hacked up a quick plugin for quicklook that uses pymol to render an image of the protein structure and store it for instant retrieval at a later date.

At the moment, the rendering process is rather slow, due to limitations of the command line rendering by pymol, it enforces ray tracing and this seriously slows things down. For some structures it will take close to 9 seconds to render. I am also dealing with a bug at the moment with the osx plugin system for quicklook, which refuses to generate the previews outside of debug mode. I’m not sure how to remedy this…

I’ve got some screenshots below of the work in progress, and I might try out the plugins on lion in the coming weeks.

 

Coupled and uncoupled mitochondria

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So for a quick refresher on the Mitochondria. It is essentially a double membraned organelle with an intermembrane space – the space between the inner membrane and outer membrane – and the matrix – the space enveloped by the inner membrane.

As I previously mentioned in my cellular respiration article, the mitochondrion encompasses the tricarboxylic Acid (TCA) cycle and the electron transport chain (ETC).  The action of the TCA cycle occurs within the matrix of the mitochondrion. It uses the chemical energy from Acetyl-CoA and charges electron carrier molecules like NAD+ and FAD+.  These charged electron carriers are then taken into the complexes of the ETC to pump H+ ions across the inner membrane to the intermembrane space to form a proton gradient.  The proton gradient exists because H+ ions are unable to cross the inner membrane yet under the laws of diffusion, the molecules want to equilibrate, thus giving them potential energy.

The important complex in this case is ATP synthase which acts as a channel to allow for H+ ions in the proton gradient to equilibrate; as this occurs, free energy is harvested and ATP synthase catalyses the formation of ATP from ADP and Pi.

This is how coupled mitochondrion work. Uncoupled mitochondrion on the other hand have a leaky inner membrane, where H+ ions are able to bypass ATP synthase and diffuse through the inner membrane.  This can occur for a number of reasons.

In this post, I’d like to talk about how 2,4-dinitrophenol causes the uncoupling of mitochondria and the effects of oligomycin, an ATP synthase inhibitor on both coupled and uncoupled mitochondria.

2,4-dinitrophenol

Quite simply, 2,4-dinitrophenol (DNP) uncouples the mitochondria by shuttling H+ ions across the inner membrane, bypassing ATP synthase. DNP is a mobile ionophore, this means that it is a lipid soluble molecule that is capable of transporting ions across a biological membrane. The way it does this is by shielding the charge of the ion within its hydrophobic exterior, thereby facilitating the transport of H+ ions across a biological membrane.

This mode of action provides less resistance to the H+ ion than as if it were to move through ATP synthase and thus it becomes the preferred route. Because of this, less ATP is produced inversely proportional to the concentration of DNP. Subsequently the potential energy that was stored in the concentration gradient is released as heat energy. If the concentration of DNP becomes too great, the cell will be unable to produce ATP and eventually die.

ATP Synthase inhibition

In regular coupled mitochondria, ATP synthase is the only release of H+ ions from the proton gradient in the intermembrane space. If ATP synthase is inhibited by oligomycin for instance, H+ ions will continue to be pumped across to the intermembrane space and not be released.  Eventually the concentration of H+ ions will become so great that the free energy of the charged electron carriers (NADH, FADH2) would not be enough to continue pumping H+ ions across the inner membrane.  Subsequently, this would stop the ETC, later on the TCA cycle and glycolysis, eventually stopping ATP synthesis, thereby killing the cell.

In uncoupled mitochondria, this would not necessarily be a fatal problem because, although ATP synthesis is not occurring from oxidative phosphorylation, H+ ions are still allowed to flow back across the plasma membrane into the mitochondrial matrix, therefore not halting the ETC, TCA cycle and glycolysis. So whilst only a small amount of ATP is being generated, it will not necessarily kill the cell.

Genetic conflicts in the transition to multicellularity

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Abstract

Our understanding of the transition from unicellular life to multicellular life is not complete. There are two major models used to describe how multicellularity might develop – the colonial model and the aggregation model.

However, both models pose problems that life must overcome in order to achieve multicellularity.  For example, while complex multicellular organisms require cells to function cooperatively, cooperation invites selfish genomes to promote their own growth at the expense of the multicellular organism.  When multicellular organisms develop via the colonial model, they are protected due to lowered chances of genetic variation, as all cells are clones of a single parent cell.  In the aggregation model, cells of similar but non-identical lineages associate to form one multicellular organism.  The aggregation mode of development is the weaker of the two, as it is more susceptible to cheating genomes.

This article aims to outline the tradeoff between the benefits of multicellularity and the challenges towards such development via the two models.  Specific genetic conflicts and defector cell lineages that arise through each model are examined, and means by which lineages may overcome such hindrances are discussed.  It is argued that multicellularity is beneficial and achievable such that such life is widespread.  It has been observed that selfish genomes are not selected for and rarely survive on their own.  However, cooperative cell systems have developed a number of mechanisms to defend against selfish genomes.  It is apparent that such mechanisms are vital to the survival and evolution of complex multicellular organisms.

Introduction

Multicellularity has independently developed at least 25 times in both prokaryotes and eukaryotes (Bonner 1998, 2000; Buss 1987; Carroll 2001; Cavalier-Smith 1991; Maynard Smith & Szathmáry 1995; Medina et al. 2003). However, our understanding of the transition to complex multicellular life is incomplete. Szathmáry & Wolpert (2003) suggest that with over 20 independent occurrences of multicellularity, the transition to multicellularity must not be difficult. However, they also note that of these 25 occurrences of multicellularity, only three major lineages have produced complex organisms – plants, animals and fungi – over 3.5 billion years of evolution, and that this is perhaps a small number after all.

Thus, there may be developmental or genetic ‘hurdles’ to overcome before multicellularity can persist. Despite this, multicellular organization is frequent and wide spread amongst life, suggesting that this type of life provides some selective advantages over unicellular life (Bonner 2000; King 2004; Medina et al. 2003). The hurdles towards multicellularity might include genetic conflicts. For instance, all cells within a multicellular organism must cooperate; however, if a cell or group of cells becomes selfish and squanders resources or produces chemicals that yield detrimental results to the organism, the overall fitness of the organism will be reduced, and the selfish cell will not propagate (Burt & Tivers 2006; Dawkins 1976; Hamilton 1964a, 1964b; Maynard Smith, & Szathmáry 1995; Michod 1997, 2003).

As biological systems increase in complexity, an increase in genetic conflicts is also observed (Maynard Smith, & Szathmáry 1995; Michod & Roze 2001). How are the cells that selfishly squander the benefits of cooperation kept at bay so that the increase in complexity becomes established throughout the population at the species level? In this article, I aim to investigate the conflicts that selfish genomes impose on the transition from unicellular to multicellular life, and discuss ways in which such conflicts are resolved so that the transition may become established. I propose that one of the less understood hurdles may include genetic conflicts.  I will also discuss the different modes of multicellular development.

Genetic conflict in the development of multicellular life

The transition from unicellular to multicellular organization may be relatively easy in developmental terms.  Many of the requirements for multicellular organization including cell-cell signaling, cell adhesion and programmed cell death were most likely already present in unicellular ancestors (Bonner 2000; Hynes & Zhao 2000; King et al. 2003; King 2004; Koonin & Aravind 2002; Miller & Bassler 2001).  According to King (2004), there are two generalized approaches to the transition to multicellularity: colonial development and aggregative development.  Multicellularity ultimately depends upon the cooperation amongst cells; however, cooperation provides the opportunity for selfish genes to take hold at the expense of the organism (Michod 2003).

Figure 1: Multicellular development modes

 

Colonial development model

Colonial development occurs when a unicellular spore or zygote divides and the cells stay attached to or encased within one another (Bonner 2000; King 2004).  According to Bonner (1998, 2000), this type of development is observed in almost all multicellular aquatic and terrestrial organisms.  In the colonial development model, genetic conflict is relatively low. Genetic variation between the cells of an organism is minimal due to the organism arising from a single cell.  Mutations within cell lines usually arise through somatic mutation and pathogen infection (Dawkins 1976; Hamilton 1964b; Michod & Roze 2001).  Genetic variation, unless occurring in germ cells, is generally not passed on to offspring.  It should be noted that cellular specialization of the colonial model is caused by changes in gene expression or post-transcriptional modifications and not by genetic variation (King 2004).

Defectors in the colonial development model are generally cancerous in nature (Frank 2003).  Cancers are the most obvious example of defecting cell lines, as they display uncontrolled cellular growth at the expense of the organism (Frank 2003).  Frank (2003) suggests that cancers rarely take hold of somatic tissues and thus are rarely transmitted across generations.  However, cancerous cell lines do not hold any evolutionary advantage; in fact, they disadvantage the organism and thus rarely survive beyond the life span of the organism (Frank 2003).  Strathmann (1991) indicates that any obligate defector – one that rapidly divides and fails to conform to the normal order of an organism – is unlikely to survive on its own.

Failure for such a defector to survive would be due to its nutrient requirements, where such requirements cannot be procured without the help of other cells (Strathmann 1991).  This is supported by Fiegna & Velicer (2003), who found defector strains of Myxococcus xanthus could only persist in the presence of normal strains.  The defector strains were then isolated, produced abnormal multicellular structures and ended in self-extinction.  Defector cell lines that exist within multicellular organisms of colonial development will in most cases serve to damage the organism and are rarely passed on through to offspring.  Defector cell lines are at an evolutionary disadvantage and in most cases are unable to survive on their own.

Aggregative development model

Aggregative development occurs when cells living independently associate with one another to become a multicellular organism (Bonner 2000; King 2004).  This mode of development is most common amongst cellular slime molds, myxobacteria, myxomycetes and some ciliates (Bonner 2000).  For example, cellular slime molds will generally live an individual unicellular life, but when starved will associate with one another to form a multicellular body (Bonner 2000; Hudson et al. 2002; King 2004).  The body will consist of sterile stalks that support the fertile mass of spores.  These sterile stalks are sacrificed to ensure that the spores are able to increase their chances of propagation (Hudson et al. 2002). In this case, it is not uncommon for cheating to occur. This might involve cells unrelated to the altruistic stalks using their physical structures to better their dispersal and reproduction without giving anything in return (Hudson et al. 2002).

According to Bonner (2000), multicellularity occurs more rarely through aggregative development than through colonial development.  This is because aggregative development can easily involve cells of different genetic lineages.

In addition, due to the greater chances of genetically different cells being incorporated into the multicellular organism, the aggregation model increases the potential for cheating cell lineages to survive, spread and persist in populations (Hamilton 1987a,b; Maynard Smith & Szathmáry 1995; Michod & Roze 1999). Therefore, the increased levels of cheating in the aggregative model causes it to be weakly selected for when compared to the colonial development model. As such, aggregative development is less prevalent amongst multicellular life than colonial development.

 

Defences against genetic conflicts
Michod (2003) defined two types of conflict mediators that are used to repress the effects of defectors.  The first mediator limits the survivability of defectors in the sense that they will be restricted in spreading throughout a population and is generally limited to the lifespan of the multicellular organism; this is a group level mediator.  The second mediator includes a number of mechanisms such as germ cell segregation, programmed cell death and self/non-self recognition systems.  These mediation systems are used to resolve intra-organism conflicts and operate on an organism level.

Germ cell segregation

Michod (1997, 2003) defines germ cell segregation as the ability for a multicellular organism to separate one cell or a group of cells aside from the rest to be used as the reproductive cell or cells of that organism.  This provides protection in that if mutations or defectors arise within the somatic cell lines of the organism, the mutations or defectors are not transmitted to subsequent generations during reproduction as the germ cells remain unaffected.  Because of the protection this provides to the organism, germ cell segregation is one of the key protection mechanisms available to complex multicellular organisms (Buss 198a,b, 1987; Michod 1997, 2003; Michod & Roze 2001).

Programmed cell death

Koonin & Aravind (2002) describe programmed cell death (PCD) as a number of mechanisms that are involved in cellular suicide under a range of different conditions. Koonin & Aravind (2002) stated that whilst PCD is universally present, a more commonly known system, apoptosis, is limited only to eukaryotes.  Apoptosis is the most widely known programmed cell death systems, in which a cell will destroy its self in response to external or internal stimuli and does so in a way to minimize the amount of damaging materials released (Lodish et al. 2000).  According to Meier et al. (2000) apoptosis is essential to the development of multicellular organisms, especially in the maintenance and repair of tissues.  The ability to induce the death of misplaced and or damaged cells is vital to the survival of any complex multicellular organism.

In a study of three organisms – the nematode, fruitfly and mouse – disruption in the process of apoptosis resulted in a large number of different developmental problems (Meier et al. 2000).  Michod (2003), supported by Meier et al. (2000) and Hamilton (1964a,b), also argues that programmed cell death has a role in mediating genetic conflicts within an organism.  According to Kaufmann & Gores (2000), one of the most commonly observed defectors within colonial based multicellular organisms, cancers, generally prevent or disrupt apoptosis.  Although apoptosis is not used to necessarily destroy cancerous cell lines, PCD is still effective in destruction of mutant or damaged cells and demonstrates a method for regulation of development of a multicellular organism.

Self/non-self recognition

Grosberg (1988) defines self/non-self recognition as the ability for a multicellular organism to use genetic, environmental and or physiological mechanisms to differentiate cells or tissues as self or non-self.  Such an ability to recognize defector cells provides the multicellular organism with an increased defensive capability against selfish genotypes.

Nearly all multicellular organisms posses the capability to recognize self from non-self (Grosberg 1988). For instance, the vertebrate immune system is based entirely on the ability to differentiate self from non-self and does so extremely well (Frank 2003).  Overall, a self/non-self recognition system is essential to the detection and removal of non-cooperative cell lines amongst multicellular organisms.

Conclusion

It has been observed that the colonial developmental model is inherently more protective against selfish genomes compared to the aggregative model (Bonner 1998, 2000; King 2004; Michod 2003).  When multicellular organisms develop via the colonial model, they are protected due to lowered chances of genetic variation, as all cells are clones of a single parent cell.  In the aggregation model, cells of similar but non-identical lineages associate to form one multicellular organism.  The aggregation mode of development is the weaker of the two, as it is more susceptible to cheating genomes.  This is supported by the large adoption to the colonial model amongst multicellular life on the planet (Bonner 1998, 2000; King 2004).

Genetic conflicts and defector cell lineages that arise through each model have been examined, and means by which lineages may overcome such hindrances are discussed.  It is argued that multicellularity provides a strong selective advantage over unicellular organisms and must be readily achievable due to the large abundance of multicellular life.  It has been observed that selfish genomes are not selected for and rarely survive on their own.  However, cooperative cell systems have developed a number of mechanisms to defend against selfish genomes (Dawkins 1976; Michod 2003).  It is apparent that such mechanisms are vital to the survival and evolution of complex multicellular organisms.

References:

Burt A, Trivers R. 2006. Genes in conflict: The biology of selfish genetic elements. Cambridge, MA: Harvard University Press. 602 pp.

Bonner JT 1988. The evolution of complexity by means of natural selection. Princeton, NJ: Princeton University Press. 272 pp.

Bonner JT. 1998. The origins of multicellularity. Integr. Biol. 1:28-36

Bonner JT. 2000. First Signals: The evolution of multicellular development. Princeton, NJ: Princeton University Press. 146 pp.

Buss LW. 1983a. Evolution, development and the units of selection. Proc. Natl. Acad. Sci. USA 80:1387-91

Buss LW. 1983b. Somatic variation and evolution. Paleobiology 9:12-16

Buss LW. 1987 The evolution of individuality. Princeton, NJ: Princeton University Press. 201 pp.

Carroll SB. 2001. Chance and necessity: the evolution of morphological complexity and diversity. Nature 409:1102-9

Cavalier-Smith T. 1991. Cell diversification in heterotrophic flagellates. In the Biology of Free-living Heterotrophic Flagellates, ed. DJ Patterson, J Larsen, pp. 113-31. Oxford, UK: Clarendon.

Dawkins R. 1976. The Selfish Gene. Oxford, UK: Oxford University Press.

Frank SA. 2003. Repression of competition and the evolution of cooperation. Evolution 57:693-705

Fiegna F, Velicer GJ. 2003. Competitive fates of bacterial social parasites: persistence and self-induced extinction of Myxococcus xanthus cheaters. Proc. R. Soc. London Ser. B 266:493-98

Grosberg RK. 1988. The evolution of allorecognition specificity in colonal invertebrates. Q. Rev. Biol. 63:377-412

Hamilton WD. 1964a. The genetical evolution of social behaviour. Part I. J. Theor. Biol. 7:1-16

Hamilton WD. 1964b. The genetical evolution of social behaviour. Part II. J. Theor. Biol. 7:17-52

Hudson RE, Aukema JE, Rispe C, Roze D. 2002. Altruism, cheating, and anticheater adaptions in the cellular slime molds. Am. Nat. 160:31-43

Hynes RO and Zhao Q. 2000. The evolution of cell adhesion. J. Cell Biol. 150(2):F89-F95

Kaufmann SH, Gores GJ. 2000. Apoptosis in cancer: cause and cure. BioEssays 22:1007-17

King N. 2004. The unicellular ancestry of animal development. Dev. Cell 7:313-25

King N, Hittinger CT and Carroll SB. 2003. Evolution of key cell signaling and adhesion protein families predates animal origins. Science 301:361

Koonin EV, Aravind L. 2002. Origin and evolution of eukaryotic apoptosis. Cell Death Differ. 9:394-404

Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell J. 2000. Molecular Cell Biology. New York: Freeman. 4th ed.

Maynard Smith J, Szathmáry E. 1995. The Major Transitions in Evolution. Oxford University Press. 346 pp.

Meir P, Finch A, Evan G. 2000. Apoptosis in development. Nature 407:796-801

Medina M, Collins AG, Taylor JW, Valentine JW, Lipps JH, Amaral-Zettler L and Sogin ML. 2003. Phylogeny of Opisthokonta and the evolution of multicellularity and complexity in Fungi and Metazoa. Int. J. Astrobiol. 2:203-11

Michod RE. 2003. Cooperation and conflict during the origin of multicellularity. Cambridge, MA:MIT Press.

Michod RE, Roze D. 2001. Cooperation and conflict in the evolution of multicellularity. Hereditary 86:1-7

Michod RE. 1997. Cooperation and conflict in the evolution of mutlticellularity. I. Multilevel selection of the organism. Am. Nat. 149:607-45

Miller MB, Bassler BL. 2001. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55:165-99

Pál C, Papp B. 2000. Selfish cells threaten multicellular life. Trends Ecol. Evol. 15:351-52

Raff RA. 1988. The selfish cell lineage. Cell 54:445-46

Sinervo B, Chaine A, Clobert J, Calsbeek R, Hazard L, Lancaster L, McAdam AG, Alonzo S, Corrigan G, and Hochberg ME. 2006. Self-recognition, color signals, and cycles of greenbeard mutualism and altruism. Proc. Natl. Acad. Sci. USA 103(19): 7372–7377

Strathmann RR. 1991. From metazoan to protest via competition among cell lineages. Evol. Theory 10:67-70

Szathmáry E, Wolpert L. 2003. The transition from single cells to multicellularity. As part of Hammerstein’s Genetic and cultural evolution of cooperation. MIT Press. 301 pp.

Velicer GJ, Kroos L, Lenski RE. 1998. Loss of social behaviours by Myxococcus Xanthus during evolution in an unstructured habitat. Proc. Natl. Acad. Sci. USA 95:12376-80

Cellular respiration

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As I mentioned in my first post, I was working on a nice explanation for cellular respiration so here it is. I’ll try to keep it as simple as possible but keep all the information there, however this is mostly aimed at second/third year university students and anyone who might want a refresher.

What is cellular respiration?

Cellular respiration is quite simply a series of reactions that converts glucose in the presence of oxygen into ATP (Adenosine triphosphate). ATP is an energy rich molecule that is heavily used by nearly every single reaction within the cell. ATP has two forms that we will investigate here, ATP (Adenosine triphosphate) and ADP (Adenosine diphosphate). Both forms consist of adenosine, which is an adenine ring and a ribose sugar attached to phosphate groups, this is where they differ. ATP has three phosphate groups and ADP has two phosphate groups.

This is particularly useful because ATP can undergo a type of chemical reaction called hydrolysis which will remove a phosphate group and release useful energy.

ATP + H2O → ADP + Pi — ΔG˚ = −30.5 kJ/mol

It can be further hydrolysed to AMP (Adenosine monophosphate) and release some more energy.

Without going off on a tangent, cellular respiration converts glucose into ATP molecules and does so in this simplified reaction:

C6H12O6 + 6O2 → 6CO2 + 6H2O + 34 or 38 ATP

1 Glucose + 6 Oxygen → 6 Carbon dioxide + 6 Water + 34 or 38 ATP

This is considered to be a redox reaction as glucose is oxidised (loses H+ ions) into carbon dioxide and the oxygen is reduced (gains H+ ions) into water.

However like most things, cellular respiration is much more complex than is suggested by that formula. This diagram shows a very nice overview of the entire system.

Cellular respiration diagram

So cellular respiration can be broken down into 4 major steps:

  1. Glycolysis.
  2. Formation of Acetyl Co-enzyme A.
  3. TCA cycle/Kreb’s cycle/Citric acid cycle.
  4. Electron transport chain/Oxidative phosphorylation.

Each stage has its own location in which it occurs. This can be seen in the diagram but I’ll go into more detail on this.

Glycolysis

Glycolysis is the first stage of cellular respiration and occurs in the cytoplasm, its overall reaction converts 1 molecule of glucose into 2 molecules of pyruvate and yields 2 molecules of ATP and 2 molecules of NADH. Glycolysis can be further broken down into two stages. I’ll talk about the importance and role of NADH in the electron transport chain section.

In pretty much all texts, glycolysis is broken down further into two stages, the energy investment stage and energy harvesting stage. Glycolysis is considered to be the root metabolic pathway of a lot of living organisms with some slight variations between aerobic and anaerobic organisms. Because of the high occurrence of glycolysis amongst prokaryotes, eukaryotes, anaerobes and aerobes, it is believed that glycolysis is one of the most ancient metabolic pathways. Glycolysis does not require oxygen to proceed and is a feeder for a number of different pathways like ethanol fermentation and lactic acid fermentation.

In the energy investment stage, we use 2 molecules of ATP and convert glucose into fructose 1, 6-biphosphate. Fructose 1, 6-biphosphate is now essentially split it in half forming two molecules of glyceraldehyde 3-phosphate or G3P for short.

The energy harvesting stage picks up G3P and breaks it down further into pyruvate through a series of reactions. Each series is done twice and each yields 2 molecules of pyruvate, 2 molecules of ATP and one molecule of NADH.

This gives a total of 4 ATP, 2 NADH and 2 pyruvate molecules. However the investment stage required the use of 2 ATP molecules, thus we get a net yield of 2 ATP, 2 NADH and 2 pyruvate molecules for the entirety of glycolysis.

Reaction summary:

Glucose + 2 NAD+ + 2 Pi + 2 ADP → 2 pyruvate + 2 NADH + 2 ATP + 2 H+ + 2 H2O


Formation of Acetyl Co-enzyme A

This is the second stage and it picks up from the path of glycolysis where pyruvate is converted into Acetyl Co-enzyme A through a process called pyruvate decarboxylation. This occurs in the mitochondria in the presence of pyruvate dehydrogenase.

So basically what is happening here is the two pyruvate molecules are transported into the mitochondrial matrix, pyruvate is decarboxylated (having a CO2 molecule removed) and NAD+ molecule is reduced to NADH as a product of removing CO2. This now 2 carbon molecule is attached to Co-enzyme A to form Acetyl Co-enzyme A.

This is a yield of 2 Acetyl CoA, 2 CO2 and 2 NADH molecules. Now we are ready to use the Acetyl CoA molecules in the Kreb’s cycle.

Reaction summary

2 pyruvate + 2 NAD + 2 CoA → 2 Acetyl CoA + 2 NADH + 2 CO2

The Krebs Cycle



I wanted to show you a simplified diagram of the Krebs Cycle before I dabble into what it is and it’s primary function as I think it might help to visualise the cycle. So basically the Krebs cycle/Citric Acid Cycle/Tricarboxylic Acid Cycle is a series of enzyme catalysed reactions that occur within the matrix of the mitochondria. It is an aerobic pathway that is used to break down carbohydrates, fats and proteins into carbon dioxide, water and useable energy in the form of NADH and FADH2 and ATP.

The Krebs Cycle contains 8 stages where by a specific enzyme catalyses each reaction and a lot of the compounds and molecules are recycled and reused over and over again.

The first reaction involves acetyl CoA transfers its two carbon acetyl group to the four carbon acceptor oxaloacetate, thus forming citrate, a six carbon compound. This releases the CoA component of acetyl CoA and is subsequently reused in acetyl CoA formation.

Citrate then moves through a series of transformations, losing one and then two carboxyl groups as CO2. This happens in the steps between citrate and ketoglutarate, and between ketoglutarate and succinate. CO2 is discarded as waste. One ATP is formed per acetyl group, however most of the energy is available as energy-rich electron carriers like NAD+ and FAD. For each acetyl group that enters the cycle, 3 molecules of NADH, 1 molecule of FADH2 and 1 molecule of ATP are produced.

Because 2 acetyl CoA molecules are produced from each glucose molecule, we account for two cycles, thus yielding a total of:

  • 4 CO2
  • 6 NADH
  • 2 FADH2
  • 2 ATP

At this point in aerobic respiration, we have only produced 4 molecules of ATP, which is pretty minimal for such a complex process of oxidising two simple acetyl groups into CO2. This seems quite inefficient, but the Kreb’s cycle is an intermediate metabolic pathway that can process a wide number of 4 and 5 carbon molecules to produce fuel. In some instances the metabolites can be drawn out of the cycle to be used in other metabolic pathways. Evolutionarily speaking, whilst this is not the shortest path of acetate to CO2, being able to process a wider number of molecules has proven to have the greatest selective advantage.

With only 4 ATP so far, where does all the energy come from?

You know all those electron acceptors I’ve been talking about, NAD+ and FAD. Well their importance will finally be revealed in the next and final stage, the electron transport chain.

Reaction summary

2 Acetyl CoA + 6 NAD+ + 2 FAD + 2 ADP + 2 Pi + 2 H2O → 4 CO2 + 6 NADH + 2 FADH2 + 2 ATP + 2 CoA

For a more detailed image click here.

The electron transport chain

Before I start on the electron transport chain, I’d like to give a brief introduction into what electron carriers are, their function and importance. Firstly some definitions.

  • A molecule that accepts an electron is reduced.
  • A molecule that loses an electron is oxidised.

Electron carriers are essentially molecules that can easily accept or donate electrons depending on the reducing power interaction between the electron carrier and a donor or acceptor. What I mean by this is that if NADH, a charged electron carrier were to come in contact with another agent of higher reducing power, it will become oxidised and revert to its uncharged state NAD+. On the other hand if NAD+ comes in contact with an agent of lower reducing power NAD+ will become oxidised and then become NADH again.These molecules can happily revert between the two without being consumed.

Electron carriers like NADH and FADH2 are not only used in aerobic respiration but act as substrates for various ligases, coenzymes and intermediate signalling molecules.

So the electron transport chain is my favourite part of aerobic respiration because of the neat way in which it works. It is essentially consists of 4 protein complexes that reside in the inner membrane of the mitochondria, these complexes are involved with carrying electrons from one complex to another due to the redox potential of each complex. In doing so the electrons loose a small amount of energy as they move through the chain.

The chain consists of these 4 complexes plus a 5th unlinked complex, ATP synthase. Three of the 4 complexes act as pumps, moving H+ ions across from the matrix into the intermembrane space. This builds up a concentration gradient on one side that wants to equilibriate with the matrix. So this is creating an electrochemical force that can be utilised as energy by ATP synthase.

Complex I: (NADH – Ubiquinone oxidoreductase) accepts electrons from NADH that have been produced previously by glycolysis, the formation of acetyl CoA and the TCA cycle. It then passes these electrons directly to Ubiquinone (Q). At the same time, 4 H+ ions are pumped across to the intermembrane space. As these electrons have been transferred to Q, it is now in the reduced for of QH2 and can freely move out of complex I and over to complex III.

NADH + 5 H+(negative side) + Q →  NAD+ + QH2 + 4H+(positive side)

Ubiquinone or Q is a lipid bound electron carrier, it provides an intermediate shuttle of electrons between complexes in hydrophobic environments. Because it is both small and hydrophobic, it can freely move within the inner membrane to carry electrons between other membrane bound complexes. It can be reduced and accept one electron to become (QH) or two electrons and become (QH2).

Complex II: (Succinate – Ubiquinone oxidoreductase) accepts electrons from FADH2 molecules that were produced during the TCA cycle. Like complex I, it produces reduced Q as QH2. The reduced QH2 is now free to move to complex III, however unlike complex I, it does not act as an electron pump.

Complex III: (Ubiquinone – Cytochrome c oxidoreductase) accepts reduced QH2 from complexes I and II and passes electrons to cytochrome c. This process is quite complex and is out of the scope of this article. But the overall net effect is quite simple. QH2 is oxidised to Q and two molecules of cytochrome c are reduced. In this case, cytochrome c is used as an electron carrier that moves from complex III to complex IV once reduced. At the same time 4 H+ ions are pumped across the membrane.

Complex IV: (Cytochrome c oxidase) in a nut shell accepts electrons from Cytochrome c and uses it to reduce molecular oxygen to water. At the same time, complex IV can use the residual energy from this reduction to pump 2 H+ ions across the inner membrane. Because oxygen is the last electron acceptor in the chain, if it is not present, each acceptor molecule in the chain retains its electrons and this blocks the entire chain. This is why you need oxygen.

Because oxidative phosphorylation or ATP synthesis requires an electrochemical gradient of H+ ions, if the electron transport chain is blocked, the H+ concentration will eventually equilibriate and ATP synthesis will stop. Most multicellular organisms cannot live very long without oxygen because the ATP produced by glycolysis alone is insufficient to sustain life.

Lack of oxygen is not the only factor that can limit the electron transport chain, cyanide for instance binds strongly to the heme group that exists in Cytochrome c molecules, thus stopping the transport of electrons from complex III to complex IV. This eventually leads to the halt of ATP synthesis and the cell can no longer sustain its self.

Complex V: (ATP synthase) is the final stage of oxidative phosphorylation. This is where the magic happens, all the hard work put in by the other stages to pump H+ ions across a membrane to create a electrochemical gradient is finally payed off. ATP synthase is essentially two motors an ion pump and an enzyme all wrapped together.

Motor 1 (F0) is coupled with a proton pump uses the electrochemical energy of the H+ ions from the intermembrane space to rotate, because the motors are joined via the stator, the rotation of F0 forces the rotation of F1 and thus drives the generation of ATP. I mentioned that F1 was coupled with an enzyme, this enzyme in particular is capable of phosphorylating ADP + Pi to produce ATP.

The overall mechanism is as follows, H+ ions pass through the F0 ion pump, this movement causes F0 to rotate. Because F0 and F1 are attached by the stator, rotation in F0 causes rotation in F1, F1 is coupled with an enzyme that cycles between 3 states. The “open” state where ADP and Pi can enter the active site, when this happens the enzyme changes shape and closes trapping ADP and Pi, this is called the “loose” state. Finally, the enzyme shifts into the “tight” state where it brings the ADP and Pi so close together that it forces ADP and Pi to bond and form ATP.

As F0 continues in its rotation, it allows the F1 enzyme to shift back into the open state and release ATP back into the matrix.

The number of H+ ions that are used to generate a single molecule of ATP varies between 3 and 4, and in some cases the cell can control this ratio for various reasons. In the presence of high ATP, ATP synthase can work in reverse to pump H+ ions back across the membrane.

Summary

The electron transport chain accepts electrons from NADH and FADH2 that were produced from glycolysis and the TCA cycle, it passes these high energy electrons through a series of enzyme complexes that eventually oxidise it to form water in the presence of oxygen. At the same time, the energy released from oxidisation of these molecules is used to pump H+ ions across a membrane which creates an electrochemical gradient or proton-motive force that is subsequently harnessed by ATP synthase to produce a more stable energy transport molecule that a wide range of reactions can harness.

Aerobic respiration summary table

Stage Co-enzyme yield ATP yield
Glycolysis investment -2 ATP
Glycolysis harvesting 2 NADH 4 ATP
Formation of Acetyl CoA 2 NADH
TCA cycle 6 NADH
2 FADH2		 2 ATP
Electron transport chain -10 NADH
-2 FADH2		 30 ATP
4 ATP
Total 36 to 38 ATP

References

Solomon, et al 2005. Biology, 8th edition. Thomson Brooks/Cole.

Nelson, D.L, Cox, M.M  2004. Lehninger Principles of Biochemistry, 4th edition. W. H. Freeman.

Gresser MJ, Myers JA, Boyer PD 1982. Catalytic site cooperativity of beef heart mitochondrial F1 adenosine triphosphatase. Correlations of initial velocity, bound intermediate, and oxygen exchange measurements with an alternating three-site model. J. Biol. Chem. 257 (20): 12030–8.

P. D. Boyer 1997. The ATP Synthase–A Splendid Molecular Machine. Annual Review of Biochemistry, 66: 717-749.

Welcome

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Well this is my first post, not really sure what to say here, but I’m working on a nice explanation of cellular respiration and all of the constituent processes. More to follow soon. I also partially made a video on the process which, if I get the time to finish will post on youtube. If for some reason someone actually reads this, feel free to make a suggestion as to my next article.

Cheers, Bacteria.