These materials will help you to prepare your PowerPoint presentations for BIOC 2300 seminar 1. You only have five minutes in total, so most of your presentation will inevitably be drawn from textbooks. It would be good to briefly mention current research. Concentrate on one or two key diagrams and try to identify and highlight the principal points.
Start with a recent text book, available in multiple copies from the University Library. Try to locate the latest edition since this is a rapidly changing field.
Alberts et al (2008) Molecular Biology of the Cell 5th edn. Garland Science. Read chapter 14.
Lodish et al (2007) Molecular Cell Biology 6th edn. W H Freeman. Read chapter 16.
Nelson & Cox (2008) Lehninger: Principles of Biochemistry 5th edn. W H Freemnan. Read chapter 19.
You should in any case understand the following key information, and your presentation should reflect this information where it is relevant to your allocated topic:
P:O ratios with different substrates.
distinguish "state 3" from "state 4" respiration.
magnitude and orientation of the electrical and pH gradients.
proton motive force = electrical component (150-180mV) + pH component (~0.5 pH units).
the difference between uncouplers, ionophores, respiratory chain inhibitors, phosphorylation inhibitors and transport inhibitors.
huge size, vague structure.
FMN, non-heme iron and ubiquinone.
four protons pumped per electron pair.
X-ray structure determined.
non-heme iron, cytochromes C, C1, BH and BL.
Q cycle, four protons pumped per electron pair.
X-ray structure determined.
cytochromes C, A, A3 and copper.
two protons pumped per electron pair.
X-ray structure determined.
ATP binding energy and conformational changes.
two motors coupled "back to back" with a rotating drive shaft.
approximately three protons used per ATP, PLUS another one for substrate pumping.
Many students find this calculation difficult, because the respiratory chain is "folded" across the mitochondrial inner membrane. Electrons enter the inter-membrane space at complex 3 but return to the matrix at complex 4. This implies that for each NADH molecule oxidised by the electron transport chain, complex 3 exports four protons, but only two positive charges leave the matrix space, whereas complex 4 exports only two protons but four positive charges leave the matrix space.
cytosol / intermembrane space
mitochondrial matrix space
We can draw up a detailed balance sheet for proton movements across the inner membrane and ATP synthesis.
It is apparent that each pair of electrons that travels from NADH to oxygen promotes the export of ten protons, and that four protons must re-enter the matrix space to deliver each finished ATP. Thus each NADH will support the manufacture of 2.5 ATP molecules, as is actually observed. Succinate, however, whose electrons bypass complex 1, promotes the export of only six protons, sufficient for only 1.5 ATP molecules, again as actually observed.
Click the links below to download copies of the original papers. Some of these are "open access" journals, but "subscription" journals may require you to log in via a University server.
Covian & Trumpower (2008) Regulatory interactions in the dimeric cytochrome bc1 complex: The advantages of being a twin. BBA - Bioenergetics 1777(9), 1079-1091 .
Cross (2004) Molecular Motors: Turning the ATP motor. Nature 427, 407-408.
Hunte et al (2003) Protonmotive pathways and mechanisms in the cytochrome bc1 complex. FEBS Letters 545(1), 39-46.
Senior & Weber (2004) Happy motoring with ATP synthase. Nature Structural & Molecular Biology 11, 110 - 112.
Shimokata et al (2007) The proton pumping pathway of bovine heart cytochrome c oxidase. PNAS 104(10), 4200-4205.
Zickermann et al (2008) Challenges in elucidating structure and mechanism of proton pumping NADH:ubiquinone oxidoreductase (complex I). J Bioenerg Biomembr 40, 475–483.
X-ray structures are available for ATP synthase, cytochrome oxidase (complex 4), ubiqinol:cytochrome c reductase (complex 3) and cytochrome c. Complex 1 is so big that it has so far defied all attempts to crystallise it. You can download the data files and display them using free software such as the MDL CHIME plug-in for web browsers, or Cn3D from NCBI. (Both plug-ins are pre-loaded on the University of Leeds desktop.) A wide range of Java applets now provide similar features. We have been assured that it is possible to embed some of this functionality into Powerpoint presentations, although we have yet to see this done!
Here is the X-ray structure of bovine mitochondrial ATP synthase, as reported by Abrahams et al (1994) Nature 370, 621. [Protein Data Bank code 1BMF.]
This is an interactive display using the CHIME software. Rotate the enzyme by dragging it with the left mouse button. Change its appearance by clicking the image with the right mouse button to show the structure more clearly.
Using the right mouse button, set "Display" to cartoons.
You should now be able to see nucleotides in the active sites.
Rotate the ensemble with the left button, and experiment with different views to see how the protein grips its ligands. Click HERE for a brief reminder of the main CHIME commands, or HERE for a full tutorial. Use the browser back button to return to the lecture notes.
In year two we do not normally expect you to read many original papers, however the very best are difficult to resist. Noji's group at the University of Osaka are brilliant experimentalists, and their most recent paper [Okuno et al, 2008] is not too difficult to understand:
Noji et al (1997) Direct observation of the rotation of F1-ATPase. Nature 386, 299–302. This is the classic Noji paper, now unfortunately only available as a static PDF file. Their original article included a video, which is now quite difficult to find. These images of the rotating actin filament were recorded with a fluorescence microscope in the presence of ATP. The actual motor is much too small to see.
These workers discarded the F0 basepiece and tethered the F1 motor head groups to a glass plate using polyhistidine tags attached to the N-termini of all three beta subunits. The glass plate had been pre-treated with horseradish peroxidase conjugated with the nickel complex of nitrilotriacetic acid, to which polyhistidine binds with high affinity. [Nitrilotriacetic acid looks like half an EDTA molecule, so it leaves un-coordinated nickel positions available for external ligands.]
The motors were glued down by their large catalytic subunits, leaving the motor shafts exposed, and facing away from the glass. The gamma subunits which form the shaft were modified by site directed mutagenesis to remove the original Cys193 (which is inconveniently far down the shaft) and replace it with serine. These workers also replaced Ser107 in the stalk region with cysteine.
This single cysteine residue (the only one in the molecule) could then be biotinylated, and linked using streptavidin to fluorescently labelled, biotinylated actin filaments. [Streptavidin has four biotin binding sites.]
The fluorescent actin filaments were many times larger than the tethered motors and could be visualised in a light microscope.
Addition of 2mM ATP caused a small number of motor shafts marked by the actin filaments to rotate in a counter-clockwise direction. The movie shows the results they obtained.
Circular motion also occurs in the proteins which rotate bacterial flagella, another important enzyme system which is driven by the proton motive force.
click the arrow on the
left to start the movie
Diez et al (2004) Proton-powered subunit rotation in single membrane-bound F0F1-ATP synthase. Nature Structural and Molecular Biology 11, 135 - 141.
Itoh et al (2004) Mechanically driven ATP synthesis by F1-ATPase. Nature 427, 465-468.
Okuno et al (2008) Correlation between the conformational states of F1-ATPase as determined from its crystal structure and single-molecule rotation. PNAS 105(52), 20722-20727.
These reviews are much more detailed than you need, but might be useful for checking specific points. The article by Hosler et al covers the entire respiratory chain.
Acín-Pérez et al (2008) Respiratory Active Mitochondrial Supercomplexes. Molecular Cell 32(4), 529-539.
Brandt (2006) Energy Converting NADH: Quinone Oxidoreductase (Complex I). Annual Review of Biochemistry 75, 69–92.
Crofts (2004) The Cytochrome bc1 Complex: Function in the Context of Structure. Annual Review of Physiology 66, 689-733.
Hosler et al (2006) Energy Transduction: Proton Transfer Through the Respiratory Complexes. Annual Review of Biochemistry 75, 165-187.
Hüttemann et al (2007) Regulation of mitochondrial oxidative phosphorylation through cell signaling. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1773(12), 1701-1720.
Lenaz et al (2007) The role of Coenzyme Q in mitochondrial electron transport. Mitochondrion 7, Supplement 1, S8-S33.
Wikstrom & Verkhovsky (2007) Mechanism and energetics of proton translocation by the respiratory heme-copper oxidases. BBA - Bioenergetics 1767, 1200–1214.