Bioenergetics Flashcards
At rest how much ATP does the body make a day?
1/2 your body weight. With normal amounts of work your full body weight and with heavy work one tonne.
What does chemiosmosis involve?
One or more energy release reactions. Redox creates a transmembrane gradient used to make ATP and ATP can make an energy gradient.
What are the two main sources for the Proton Motive Force?
Respiration and photosynthesis.
What are the three main uses of PFM?
ATP synthesis.
Motility.
Soulte transport.
What is the central dogma of energy conversion?
‘Release, transfer, trapping.’
Redox… transmembrane gradient…. ATP synthesis.
What step of the central drogma of energy conversion is reversible?
Transmembrane gradient…. ATP synthesis.
What step of the central dogma of energy conversion is not easily reversible?
Redox… transmembrane gradient.
How can a cell membrane resit osmosis?
If work is put in to push in the opposite direction.
What does a ‘leaky membrane’ result in?
The movement of some H+ through the membrane without any useful energy being produced.
Why do the outer membrane in bacteria and mitochondria not use chemiosmosis?
They have holes in them.
Where is the N phase found in regards to a cell?
Outside and inside an organelle.
Where is the P phase found in regards to a cell?
In the cytoplasm.
What two things can cause the breakdown of the cytoplasmic membrane?
Lsyzome and osmotic shock.
What type of bacterium does the P and N phase apply to?
Intact bacteria.
In gram negative bacteria what phase is the periplasm considered as?
P.
In what phase is ATPsynthase normally found?
N.
After osmotic shock and lyzosome activity where is the ATPase found?
Right-side-out of vesicle (N).
After the french press where is the ATPase found?
Outside out (O).
What are Mitchells 4 postulates?
- Respiratory and photosynthetic electron transfer should translocate protons.
- The ATPase should function as a reversible proton translocating ATPase.
- Energy transducing membranes should have a low effective proton conductance.
- Energy transuding membranes should posses specific exchange carriers to permit metabolites to penetrate and osmotic stability to be maintained at a high energy potential.
Why did measuring the driving force allow?
- Quantitative approach allowing calculation away from equilibrium.
- The replacement of the wrong idea about a high energy intermediate.
- Eliminates thermodynamically imposible reactions.
Energy from ATP does not come from the high energy phosphate bond. Where does the energy come form instead?
How far the mass action ratio is equilibrium.
What drives a reaction?
A increase in entropy.
How can you asses the entropy change of a system?
Measuring the flow of heat/ enthalpy across a system.
In photosynthesis what can Gibbs energy quantify?
Light absorption.
In a closed system if A becomes B what is the observed mass action ratio?
[B} obvs/ [A} obvs.
As the mass action ratio is displaced from K what happens to Gibbs free energy?
It increases.
What is Gibbs free energy at equilibrium?
0.
If the mass action ratio is smaller than K what is Gibbs?
Negative.
If the mass action ratio is bigger than K what is Gibbs?
Positive.
What two forces act on an ion gradient?
- Concentration gradient.
2. Electrochemical potential difference.
What is the membrane potential?
The electrochemical potential difference across the membrane.
What charge is inside the mitrochrondria/ bacterial cell and why?
H+ move out.
What is delta P?
The function of the electrochemical proton gradient. It has the units of mv and was defined by Mitchell.
How can TTP be used to measure the membrane potential?
The inside of the cell is negative and TTP has a positive charge (it is a cation). Despite being charged TTP can cross the membrane and sit in the bilayer as it is very hydrophobic. The amount of TTP that travels into the cell will determine the membrane potential. The TTP used is radiolaballed to quantify the data.
What is used to measure the pH of the membrane potential?
A weak acid such as acetate. This dissociates into H+ and A-. The A- will accumulate inside and HA will be at the same concentration on both sides.
What is special about thylakoid membranes and what does this show?
They have a massive pH difference and almost no membrane potential. delta P is the opposite to this. this shows that l is determined by the combination of the two and IF YOU MAKE ONE SMALLER THE OTHER GETS LARGER.
Delta p is a combination of the membrane potential and the pH difference. How was it shown that if you increase one the other will decrease?
K+ was added to reduce the membrane potential the pH difference goes up.
Membrane potential and ph difference are defined as P phase minus N phase. Which is positive and which is negative?
Membrane potential is positive as positive outside and pH diff is negative as acidic outside.
What do classic uncouplers do?
They uncouple electron transfer by carrying H+ across the membrane and not through ATP synthase. Causes energy to be wasted as heat.
FCCP/ CCP and 2-4DNP are classic uncouplers. What else are they?
Catalysts as they never get used up. This means that you only need them in small amounts.
What uncoupler is toxic and can be used as a slimming agent?
2-4 DNP.
Classic uncouplers move H+ across the membrane. What does the other category of uncouplers do to uncouple electron transfer?
They distrupt the ATP synthase so H+ can not be translocated.
What technique can be used to determine the bacterial H+/O ratio?
The oxygen pulse/ proton pulse technique.
What are the four steps of the oxygen pulse/ proton pulse technique?
- Oxidisable substrate is added to the chamber, all oxygen in the chamber is used and the bacteria become anerobic.
- Air saturated with a known oxygen medium. This causes the pumping go H+ ions and a drop in pH.
- Oxygen is exhausted and pH transient decays as the H+ leaks back in. This can be accelerated by the use of FCCP.
- Calibration with anerobic HCL occurs.
What two things does the buffer used in the oxygen pulse/ proton pulse contain and why?
Valinomycin and K+. V moves K+ across the membrane so the charge balance does not change throughout the experiment and the full extend of H+ movement can be measured.
Genes that encode ATPase are all similar in size. True or false?
False, they are all very different and can range between 79-500 amino acids.
What genes products make up the F0 domain of ATPase?
A, C, B
What gene products make up the F1 domain of ATPase?
Delta, alpha, gamma, betta, epsilon.
What did ATP genes used to make called?
unc genes (as discovered through uncoupled mutants.)
Are more genes for ATPase encoded in the mitochondria or the chloroplast?
Chlorplast. Only two genes (8,6) are now encoded in the mitochondria.
What domain of the ATPase is found in the membrane?
F0 (named after the biding drug oligomycin)
What portion of the ATPase is catalytic?
F1.
What subunits of the ATPase make up the F1 domain?
Alpha, beta, gama, delta, eplison.
What subunits of the ATPase make up the F0 domain?
A, C, B.
What subunit of the ATPase can you recover a crystal structure of?
F1.
How many subunits of C can be present?
6-10.
What two subunits go through the middle of AB in the F1 domain of ATPase?
Eplison and gamma.
What does the catalytic mechanism of ATPase PROBABLY involve?
The rotation of the gamma subunit along with rotation of the epsilon and C subunit.
How was the rotation of the gamma/ eplison and C subunit in ATPase shown?
Experimentally though uncoupled proton translocation.
What bacterium was the rotary catalysis of ATPase found in?
Thermaphillic bacterium PS3 (bacillus species).
What were the 5 steps in determining rotary catalysis in the PS3 bacillus species?
- Express genes in E.coli so the the protein has 10 terminal his residues on each B subunit.
- Purify sub complex 3a-3b-1y.
- Bind this subunit to slide coated with Ni2+- NTA.
- Bind actin filament to 1 gamma.
- Using fluorescence microscopy watch what happens when ATP is added.
When the F0 subunit is mutated ATP synthesis will not take place. True or false?
False. ATP synthesis can take place without the proton translocating F0 portion.
What is the first step in ATP synthesis?
Proton motor force drives protons through ATPase rotating the C subunits relative to the a and b subunits.
What is the second step in ATP synthesis?
Rotation is passed onto the delta and elipson subunits.
What is the third step in ATP synthesis?
Rotation of the asymmetric game subunit mechanically causes conformational changes in the alpha/beta hexamer. Each 120 degrees rotation of the gamma subunit forces 1 of 3 catalytic sites located in the alpha/beta interface into the opened conformaiton.
What is the fourth step in ATP synthesis?
Newly synthesised ATP is released and ADP/ Pi are bound instead. High affinity of the open site to the phosphate impairs rebinding of ATP and favours ADP binding.
What is the fifth step in ATP synthesis?
The gamma subunit turns another 120 degrees forcing the next site into the opened conformation, and the ADP and phosphate bound to the previous opened site are occluded and ATP synthesis takes place.
What is the sixth step in ATP synthesis?
ATP is released once the gamma subunit has made the 360 degrees turn and the site opens again.
What are the three conformations if the A/B subunit?
Open, tight, lose.
What conformation of the A/B subunit is ATP made in?
Made in tight leaves from open.
What conformational change of the A/B subunit requires energy?
LOT - TLO.
How many degrees does the A/B subunit need to turn to make 1 ATP?
120 degrees.
How many protons are needed for a complete turn of the A/B subunit?
The same number as there are C subunits.
What does the H+/ATP ratio show?
How many hydrogens are required to make 1 ATP.
What is the H+/ATP ratio for bovine mitochondria?
2.7.
What is the H+/ATP ratio for yeast?
3.3.
What is the H+/ATP for chloroplasts?
4.7.
What is the H+/ATP for bacteria?
5.
What is the K value for ATP synthesis?
10^5.
What standard conditions are needed for the K value 10^5 for ATP synthesis?
pH7, 10mm Mg2+, 10mM Pi.
When ATP/ ADP ratios are ten magnitudes from equilibrium how much Gibbs free energy is needed?
57 KJ.
What is the purpose of the respiratory chain?
Oxidises reduced substrates and reduces an election acceptor.
What is the H+/O ratio?
Every 1/2 O2 6 protons are pumped.
What is the H+/ATP ratio?
3.3
What is the P/O ratio?
2.
In terms of reduction potentials what way do electrons flow?
From negative to positive.
What protein family does cytochrome c fall into?
Haem proteins.
What is unusual about cytochrome C?
It is soluble.
How large is cytrochome C?
Small 12-14D.
What shape is cytochrome C?
Square planer.
What atom is central to cytochrome C?
Fe. This cycles from Fe2+- Fe3+- Fe4+.
What coordinates the Fe atom in cytochrome C?
4 pyrrole rings each with a nitrogen.
What colour is cytochrome C?
Bright orange.
What changes when cytochrome C goes from oxidised to reduced?
Absorption spectra.
What four classes of reaction centres in the respiratory chain?
- FMN.
- Iron-Sulphur clusters.
- Haem B.
- Ubiquinone (ox)- ubiquinol (red).
What is the redox potential of Nad+- NADH?
-320mv.
What is the redox potential of H20?
+820 mv.
What is complex 1 in the respiratory chain?
NADH dehydrogenase.
What reaction centres are found in the respiratory complexes?
FMN, 8 Fe/S.
What is complex 2 of the respiratory chain?
Succinate dehydrogenase.
What is complex 3 of the respiratory chain?
Cytochrome BC1.
What is complex 4 of the respiratory chain?
2 haem groups and 3cu.
What complex do the pesticides rotenone and piericdin inhibit?
Complex 1.
What complex does the antibiotic antimycin inhibit?
Complex 3.
What complex does CN-, CO and NO inhibit?
Complex 4.
How do CN-, CO and NO inhibit complex 4?
They bind to the harm groups so there is no O reduction.
Unlike the mitochondria respiratory chain the respiratory apparatus of bacteria is highly ______. It is _____ meaning component assembly can be mixed and matched for a particular function. This system is ______. This is all controlled by circuits of gene regulation.
Flexible, modular, branched.
What does the bacteria respiratory chain consist of?
- Numerous dehydrogenase.
- Several quinone types.
- 3 oxidases.
- Numerous anaerobic reductases.
Is NDH1/ NADH dehydrogenase a proton pump in E.coli?
Yes.
What two oxidases in E.coli are not pumps but do translocate protons?
2 and 3.
What are the names of the two oxidases in E.coli that are not pumps but do translocate protons?
CydAB and AppBC.
What is the H+/0 ratio from the second and third oxidase in E.coli?
1.
The second and third oxidases in E.coli are not pumps. How many subunits are they made of and where are they found?
2 and they are found in the membrane.
The second and third oxidases in E.coli use proton translocation. What does this involve the spatial separation of?
H+ and e- from H2. (found in QH2).
Why are the second and third terminal oxidases in E.coli similar?
It is not known. Knocking out either the CydAB or AppBc gene has little effect.
What can bacteria alter to manipulate energy conversion?
The expression of certain dehydrogenase or oxidases.
What two deyhydrogenases can E.coli encode to alter energy conservation?
NDH1 and NDH2.
Does NDH1 or NDH2 pump 4H+ in E.coli?
NDH1.
What two terminal oxidases can E.coli encode to alter energy conservation?
Cytbo, Cytbd.
Does Cytb0 or Cytbd pump 4H+ in E.coli?
Cytob0.
What small bacteria has one of the highest respiratory capacities known?
Azotobacter vinelandii.
What type of aerobe is Azotobacter vinelandii?
Obligate
What is one of the main roles of Azotobacter vinelandii?
It fixes atmospheric nitrogen to ammonia through the use of the nitrogenase enzyme.
Azotobacter vinelandii is an obligate aerobe but the nitrogenase enzyme it uses is sensitive to oxygen. How does it overcome this?
The internal oxygen is removed by oxidase activity of cytochrome bc1.
What can the bd mutant of Azotobacter not do?
Grow by nitrogen fixation from the air.
What else can be driven by the proton motive force that doesn’t involve channels?
Flagella rotation.
What does H2SO4 produced by sulphur oxidising bacteria allow?
Leaching of metals from ores.
Why do bacteria that grow in a pH of 1-3 originally face a problem?
As H+ ions want to re enter the cell down the concentration gradient.
How do bacteria that grow in a low pH ensure that the H+ ions move the correct way?
The pH difference is maintained by respiration/ synthase and K+ and other positive ions are imported to balance the charge meaning H+ is not.
What type of circulation allows for alkaliphiles to live in a high pH?
Na+ circulation. These are pumped out of the cell by the respiratory chain and decarboxylases.
Do facultative, fermentable or photolithotropic bacteria use ATP for CO2 reduction in addition to using it for biosynthesis and transport?
Photolithotrophic.
Do facultative, fermentable or photolithotropic bacteria not use glycolysis to make ATP?
Photolithotrophic.
Do facultative, fermentable or photolithotropic bacteria use the hydrogen electrochemical gradient for reducing power in addition to using it for motility and transport?
Facultative.
Do facultative, fermentable or photolithotropic bacteria only use ATP for the hydrogen electrochemical gradient?
Fermentable.
Do facultative, fermentable or photolithotropic bacteria use light, H2S and reducing power to generate a hydrogen electrochemical gradient?
Photolithotrophic.
Do facultative, fermentable or photolithotropic bacteria use anaerobic redox and respiration as well as ATP to drive the hydrogen electrochemical gradient?
Facultative.
Do facultative, fermentable or photolithotropic bacteria use only ATP to drive the hydrogen electrochemical gradient?
Fermentable.
Do facultative, fermentable or photolithotropic bacteria use only the hydrogen electrochemical gradient to power ATP synthesis?
Photolithotrophic.
