Fermentation Lecture 11: 1Ll656883/Lectures/Lecture11.pdfBoth respiration and photosynthesis involve a series of oxidation-reduction (or electron-transport) reactions in which energy

Evolution of GAPDH

Fermentation 10/23/2006Lecture 11: 1

Enzyme assays

Steven Chu (朱棣文)

James A. Spudich

3. Hastings, J.W., Gibson, Q.H., Friedland, J., and Spudich, J. (1966) Molecular mechanisms in bacterial bioluminescence: On energy storage intermediates and the role of aldehyde in the reaction. In: Bioluminescence in Progress (F.H. Johnson and Y. Haneda, eds.), pp. 151-186, Princeton University Press.

2. Spudich, J.A. and Hastings, J.W. (1963) Inhibition of the bioluminescent oxidation of reduced flavin monucleotide by 2-decenal. J. Biol. Chem. 238: 3106-3108.

1. Hastings, J.W., Spudich, J.A. and Malnic, G. (1963) The influence of aldehyde chain length upon the relative quantum yield of the bioluminescent reaction of Achromobacter fischeri. J. Biol. Chem. 238: 3100-3105.

Steven ChuThe Nobel Prize in Physics 1997

10/23/2006Lecture 11: 2

Enzyme assays 10/9/2006Lecture 11: 3


What is it? Optical Tweezers use light to manipulate microscopic objects as small as a single atom. The radiation pressure from a focused laser beam is able to trap small particles. In the biological sciences, these instruments have been used to apply forces in the pN-range and to measure displacements in the nm range of objects ranging in size from 10 nm to over 100 mm.

A laser beam is focused by a high-quality microscope objective to a spot in the specimen plane. This spot creates an "optical trap" which is able to hold a small particle at its center. The forces felt by this particle consist of the light scattering and gradient forces due to the interaction of the particle with the light.

Principle of Operation: Fig 1b shows a more detailed look at how an optical trap works. The basic principle behind optical tweezers is the momentum transfer associated with bending light. Light carries momentum that is proportional to its energy and in the direction of propagation. Any change in the direction of light, by reflection or refraction, will result in a change of the momentum of the light. If an object bends the light, changing its momentum, conservation of momentum requires that the object must undergo an equal and opposite momentum change. This gives rise to a force acting on the object.

In a typical optical tweezers setup the incoming light comes from a laser which has a "Gaussian intensity profile". Basically, the light at the center of the beam is brighter than the light at the edges. When this light interacts with a bead, the light rays are bent according the laws of reflection and refraction (two example rays are shown in Fig 1b). The sum of the forces from all such rays can be split into two components: Fscattering, the scattering force, pointing in the direction of the incident light (z, see axes in Fig 1b), and Fgradient, the gradient force, arising from the gradient of the Gaussian intensity profile and pointing in x-y plane towards the center of the beam (dotted line). The gradient force is a restoring force that pulls the bead into the center. If the contribution to Fscattering of the refracted rays is larger than that of the reflected rays then a restoring force is also created along the z-axis, and a stable trap will exist. Incidentally, the image of the bead can be projected onto a quadrant photodiode to measure nm-scale displacements (see Further Reading).

When the bead is displaced from the center of the trap, what force does it feel? The restoring force of the optical trap works like an optical spring: the force is proportional to the displacement out of the trap. In practice, the bead is constantly moving with Brownian motion. But whenever it leaves the center of the optical trap the restoring force pulls it back to the center. If some external object, like a molecular motor, were to pull the bead away from the center of the trap, a restoring force would be imparted to the bead and thus to the motor. An example trace of a single kinesin motor taking 8 nm steps against a 5-pN force is shown in Fig 2.


Figure 2. Measurement of the 8-nm steps of kinesin.


DNA mechanics as a tool to probe helicase and translocase activity.

Nucleic Acids Res. 2006;34(15):4232-44. Epub 2006 Aug 25. Lionnet T, Dawid A, Bigot S, Barre FX, Saleh OA, Heslot F, Allemand JF, Bensimon D, Croquette V.

Helicases and translocases are proteins that use the energy derived from ATP hydrolysis to move along or pump nucleic acid substrates. Single molecule manipulation has proved to be a powerful tool to investigate themechanochemistry of these motors. Here we first describe the basic mechanical properties of DNA unraveled by single molecule manipulation techniques. Then we demonstrate how the knowledge of these properties has been used to design single molecule assays to address the enzymatic mechanisms of different translocases. We report on four single molecule manipulation systems addressing the mechanism of different helicases using specifically designed DNA substrates: UvrD enzyme activity detection on a stretched nicked DNA molecule, HCV NS3 helicase unwinding of a RNA hairpin under tension, the observation of RecBCDhelicase/nuclease forward and backward motion, and T7 gp4 helicase mediated opening of a synthetic DNA replication fork. We then discuss experiments on two dsDNA translocases: the RuvAB motor studied on its natural substrate, the Holliday junction, and the chromosome-segregation motor FtsK, showing its unusual coupling to DNA supercoiling.








Acetyl-CoA+ 3 NAD+ + FAD

+ GDP + Pi+ 3 H2O

3 NADH + FADH + CoA-SH + GTP

+ 3 CO2

C6H12O6 + 6O2

6CO2 + 6H2O + energy (ATP)

Because glycolysis produces two pyruvate molecules from one glucose, each glucose is processes through the kreb cycle twice. For each molecule of glucose, six NADH2+, two FADH2, and two ATP.

Fermentation 10/23/2006Lecture 11: 14

"for his contribution to the understanding of biological energy transfer through the formulation of the chemiosmotic theory"

Both respiration and photosynthesis involve a series of oxidation-reduction (or electron-transport) reactions in which energy is liberated and utilized for the synthesis of adenosine triphosphate(ATP) from adenosine diphosphate (ADP) and inorganic phosphate. These processes are usually called oxidative and photosynthetic phosphorylation. Both processes are typically associated with cellular membranes. In higher cells, they take place in special,membrane-enclosed organelles, called mitochondria and chloroplasts, while, in bacteria, both processes are associated with the cell membrane.

ATP serves as a universal energy currency for living cells. Thiscompound is split by a variety of specific enzymes and the energy released is used for various energy-requiring processes. The regeneration of ATP by way of oxidative and photosynthetic phosphorylation thus plays a fundamental role in the energy supply of living cells.

The above concepts had been broadly outlined by about the middleof the 1950's, but the exact mechanisms by which electron transport is coupled to ATP synthesis in oxidative and photosynthetic phosphorylation remained unknown. Many hypotheses were formulated, most of which postulated the ocurrence of 'energy-rich' chemical compounds of more or less well-defined structures as intermediates between the electron-transport and ATP-synthesizing systems. Despite intensive efforts in many laboratories, however, no experimental evidence could beobtained for these hypotheses. In addition, these hypotheses didnot provide a rational explanation for the need for a membrane in oxidative and photosynthetic phosphorylation.

The Nobel Prize in Chemistry 1978

the chemiosmotic theory 10/23/2006Lecture 11: 15

At this stage, in 1961, Peter Mitchell put forward his chemiosmotic hypothesis. The basic idea of this hypothesis is that the enzymes of the electron-transport and ATP-synthesizing systems are localized in the membrane with a well-defined orientation and are functionally linked to a vectorial transfer of positively charged hydrogen ions, or protons, across the membrane. Thus, electron transport will give rise to an electrochemical proton gradient across the membrane which can serve as a driving force for ATP synthesis. A requisite for the establishment of a proton gradient is, of course, that the membrane itself is impermeable to protons, which explains the need for an intact membrane structure in oxidative and photosynthetic phosphorylation.

The chemiosmotic hypothesis was received with reservation by many workers in the field which is, in a way, understandable, since it was unorthodox, fairly provocative, and based on little experimental evidence.


THE ELECTRON TRANSPORT CHAIN (ETC)


The job of mitochondria is to convert pyruvate to ATP and carbon dioxide. This is achieved by the interaction of NADH, and one Krebs cycle intermediate (succinate) with the inner mitochondrial membrane. This membrane contains five huge protein complexes, which serve to remove electrons from NADH, regenerating NAD, and in-so-doing, to generate a proton gradient across the membrane than may be used to drive ATP synthesis.


Complex I.Complex I is NADH dehydrogenase. It removes two electron from NADH, and transfers them to ubiquinone in the mitochondrial membrane. As the two electrons pass through various flavin(FMN), iron-sulfur (FeS) and quinone (UQ) centres, four protons are pumped across complex I into the inter-membrane space (per NADH). When the electrons are deposited onto UQ (ubiquinone), the UQ takes up a further two protons from the matrix side, to form ubiquinol(UQH2) (these are excluded from the pump-count for this complex). It produces 1 UQH2, per NADH oxidised. The ultimate source of the NADH and the electrons is the oxidation of ketoglutarate, malate etc in the Krebs cycle.

The ubiquinol formed feeds into a UQ 'pool' inside the membrane, and diffuses to complex III.


Complex II.Complex II is also called succinate dehydrogenase, and is the only membrane bound enzyme of the Krebs cycle. The dehydrogenation of succinatehas too small a ∆G for any H+ pumping, so this complex only generates 1 UQH2 per succinateoxidised, and pumps no protons. It gets its electrons from the oxidation of succinate only, and feeds them via flavin (FAD) and an iron-sulfur cluster into the UQH2 pool.


Ubiquinone and ubiquinolUbiquinone (UQ) ferries electrons from complexes I and II to complex III. Note the long hydrophobic chain: UQ/UQH2 can migrate actually dissolved within the membrane.

Partial reduction of UQ generates ubisemiquinone radicals (UQH·), which are very dangerous and must be rapidly reduced to UQH2.

Complexes I and II both feed into a pool of ubiquinol (UQH2) actually inside the inner mitochondrial membrane (dissolved in the fatty acid tails).


The reason semiubiquinone is dangerous is that is can generate superoxide radicals, which are hugely oxidising free-radicals.

UQH· + O2 → UQ + H+ + O2−·

Superoxide will dismutate to hydrogen peroxide.

2O2−· + 2H+ → O2 + H2O2

Hydrogen peroxide will undergo Fenton reaction with haem iron to produce hydroxyl radicals which are lethally destructive.

Fe2+ + H2O2 → Fe3+ + OH− + OH·

Mitochondria therefore contain superoxide dismutase and glutathione (GSH) peroxidase to cope with these agents of oxidative stress.

2GSH + H2O2 → GSSG + 2H2O


Complex III.This is also called cytochromereductase (or oxidoreductase). It pumps 4 H+ per UQH2 (including the two attached by complex I or II to UQ), and produces 2 cyt-cRED (reduced cytochrome-c) per UQH2 oxidised. The iron in the haem groups of b and c cytochromes goes from Fe3+ to Fe2+. The complex manages to pump 4 protons by running a nasty bit of biochemistry called the Q-cycle, which delivers the two electrons from one UQH2 to two cyt-c molecules, which only carry one electron each.


The 'Q-cycle' is a preposterously complicated way of transferring electrons from the two-electron-carrying UQH2 to the single-electron carrying cytochrome-c (cyt-c). A UQH2 gives up its protons to the IMS. One of its electrons is carried through FeS and cyt-c1 to the mobile cytochrome-c. The second of its electrons is carried through two cyt-b centres and is dumped back onto another UQ molecule to form a semiquinone radical. The same process then happens again with a further UQH2, fully reducing the semiquinone to UQH2. Note that the whole process consumes two UQH2, but generates one back, so there is a net oxidation of just one UQH2.

One electron from UQH2 is used to reduce cyt-c, the other is used to half-reduce a UQ in the membrane to semiquinone. This is accompanied by the release of two protons per UQH2 into the IMS. One electron from a second UQH2 is used to reduce cyt-c, and the other is used to regenerate a UQH2 in the membrane from the semiquinone radical produced earlier, with uptake of protons from the matrix. This is again accompanied by the release of two protons per UQH2 into the IMS. In upshot, only one net UQH2 has been oxidised, but four protons have been pumped.


Cytochrome b and cCytochromes are small proteins containing a haem group (much like myoglobin or haemoglobin). They are grouped into three types (a, b and c) according to the type of haemand how it is bound into the protein.

Cytochrome-b proteins contain an iron protoporphyrin-IX prosthetic group, which is bound by dipole interactions. The Fe ion is hexacoordinated: 4 ligands from the N's of haem, and 2 from the histidines in the protein.

Cytochrome-c contains a haem-c prosthetic group bound covalently by its ring to cysteinesin the protein. The Fe ion is hexacoordinated: 4 from the N's of haem, 1 from a histidine in the protein, and 1 from methionine in the protein.

Cytochrome b Cytochrome C


Complex IV.Complex IV is more commonly termed cytochrome oxidase (or even just cyt-ox). It pumps 2 H+ per 2 cyt-cRED, and produces 1 H2O per 2 cyt-cRED oxidised. Complex IV receives its electrons from cytochrome-c, which is a small, mobile protein that diffuses from complex III to complex IV. The electrons are passed through a number of cytochrome-a and copper ion centres. CuB and cyt-a3 actually perform the reduction of oxygen to water. Each NADH originally oxidisedyields 2 electrons, and these are enough to reduce half an O2 molecule to H2O (i.e. four electrons - two NADH - are required to reduce a whole molecule of dioxygen).

Cytochrome oxidase dumps electrons from cytochrome-c onto oxygen, generating water, and pumping one proton per cytochrome-c.


Cytochrome-a contains a haem-a prosthetic group bound by 'hydrophobic forces' to the protein. It also has a long phytol tail (just like chlorophyll). The Fe ion is pentacoordinated: 4 from the N's of haem, 1 from a histidine in the protein. This leaves a binding site for oxygen.


The reason that electrons flow through the various complexes is that earlier stages have lower redoxpotentials, so can provide electrons for downstream reactions.


Complex V.Complex V is ATP synthase (an F-type ATPase). It converts an H+ gradient into ATP, producing c. 1 ATP per 3 or 4 H+ (stoichiometry still not quite certain). It actually acts like a motor: the FO subunit rotates as protons flow through and ATP is synthesised due to the conformational changes this causes in F1. It probably requires 3 protons to actually form one molecule of ATP, but one further proton is required to translocate ATP out of (and ADP/phosphate into) the matrix.

As protons flow through the a/b subunits (the stator) of FO, they force the ring of twelve c subunits (the rotor) in the membrane to rotate. This rotation is transmitted to the γ/ε subunits (the stalk) of F1, which change the conformation of the α/β subunits (the headpiece) of F1, makinf ADP and phosphate react to form ATP inside the β subunits. The headpiece is prevented from rotating by the binding of δ to the a/b stator, which is itself firmly anchored in the membrane.


The electron transport chain carries the electrons produced by the oxidation of NADH to NAD through complexes I, III and IV. This electron transport is used to drive proton pumping through the membrane. The electrons are eventually dumped onto oxygen, which is reduced to water. The proton gradient built up by these processes is used to drive the FOF1 ATPase (in reverse) to generate ATP. The oxidation of 1 NADH pumps (about) 10 protons. ATPase generates (about) 1 ATP from 4 protons.

Source Anaerobic Aerobic

Glycolysis 2 ATP (substrate level phosphorylation) 2 ATP (substrate level phosphorylation)2 NADH → 0 ATP (cytosol) 2 NADH → 5 ATP

Krebs - 2 ATP/GTP (substrate level phosphorylation)- 8 NADH → 20 ATP- 2 FADH2 (succinate) → 3 ATP

Approximate total yield 2 ATP 32 ATP

NADH must be translocated into the mitochondrion from the cytoplasm: this costs ATP. The complexes are not 100% efficient. The inner membrane is not 100% impermeable. There is still argument about the stoichiometry of the various complexes. Aerobic respiration is approximately 15 times more efficient than anaerobic. The P/O ratio (ATP made per oxygen atom reduced) is about 3 for NADH and 2 for succinate (FADH2). In books, you will find many different estimates of the ATP to glucose ratio, the number of protons pumped by each complex, the proton to ATP ratio for ATPase, etc.


Chemiosmosis works by generating a proton-motive force. The proton-motive force is the free energy associated with a gradient of protons across a proton-impermeable

membrane. It is composed of two components: a chemical concentration gradient and an electrochemical charge gradient.

∆G = R T ln ( [H+]matrix ⁄ [H+]ims) − z F ∆Em


Photosynthetic archaea

Purple proteobacteria

Mitochondriachloroplasts


Science. 1999 Nov 26;286(5445):1722-4.

Mechanical rotation of the c subunit oligomer in ATP synthase (F0F1): direct observation.

Sambongi Y, Iko Y, Tanabe M, Omote H, Iwamoto-Kihara A, Ueda I, Yanagida T, Wada Y, Futai M.

Division of Biological Sciences, Institute of Scientific and Industrial Research, Osaka University, CREST (Core Research for Evolutional Science and Technology) of Japan Science and Technology Corporation, Ibaraki, Osaka 567-0047, Japan.

F0F1, found in mitochondria or bacterial membranes, synthesizes adenosine 5'-triphosphate (ATP) coupling with an electrochemical proton gradient and also reversibly hydrolyzes ATP to form the gradient. An actin filament connected to a c subunit oligomer of F0 was able to rotate by using the energy of ATP hydrolysis. The rotary torque produced by the c subunit oligomer reached about 40 piconewton-nanometers, which is similar to that generated by the gamma subunit in the F1 motor. These results suggest that the gamma and c subunits rotate together during ATP hydrolysis and synthesis. Thus, coupled rotation may be essential for energy coupling between proton transport through F0 and ATP hydrolysis or synthesis in F1.


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Fermentation Lecture 11: 1Ll656883/Lectures/Lecture11.pdfBoth respiration and photosynthesis involve a series of oxidation-reduction (or electron-transport) reactions in which energy