Poole (Biological energy transformations) Flashcards
How much ATP does an adult convert per day at diff activity levels?
- rest = 1/2 body weight ATP
- normal = body weight ATP
- hard work = up to 1 ton ATP
What did Mitchell propose?
- chemiosmotic hypothesis
- link between e- transport phosphorylation need NOT involve chemical intermediates
How does chemiosmosis underpin all contemporary thought in bioenergetics?
- redox reactions –> transmembrane grad
ATP synthesis
What is chemiosmosis?
- partially permeable membrane separates 2 solutions w/ v diff concs
- water moves through membrane to equalise concs
- at non eq distribution coupling device driven by H+ movement down ec grad and uncoupled leak of H+
- piston could be trying to push water back the way it came
How is ox and phosphorylation chemiosmotically coupled?
- resp chain extrudes protons
- gen ec pot across membrane (interior alkaline and +ve)
- return of protons through ATP synthase coupled to ATP formation
What is the structure of mito membrane?
- IM folded into cristae
- periplasm between IM and OM
What do P and N mean w/ respect to unfractionated bacterium?
- P = +ve phase, periplasm
- N = -ve phase, cyto
In fractionated bacterium what are P and N phase?
- lysozyme and osmotic shock = right side out vesicle (N inside)
- French press = inside out vesicle (P inside)
How can mito membranes be prepped?
- ultrasonication yields particles analogous to everted bacterial vesicles
In mito membranes which is P and N phase?
- P phase = IMS
- N phase = matrix
What 3 stages do all novel ideas in science pass though?
People say:
- not true
- true but not important
- true and important but not new
What is the physical reality of ‘energised state’ of, eg. a mito?
- ‘activated chemical intermediates’
- -> redox reaction creates high energy intermediate w/ assoc phosphate, but couldn’t figure out what P was
- or charged particles w/in membrane
- or pmf across membrane, ie. chemiosmosis
What are the major points of chemiosmosis?
- metabolic reactions that energise membrane gen ec grad across it
- resp chains, photosynthetic centres and ATP synthase all cat proton translocation
- polarity of pot not always +ve
- energy coupling req topologically closed membrane (vesicle)
- reagents of mutations that dissipate proton circulation also de-energise membranes
- membrane can be energised by imposing ‘artificial’ grad of pH or electrical pot
- accum of many metabolites accompanied by proton movements
How is pmf calculated?
- Δp (mV) = Δψ - 61ΔpH
- Δψ is charge diff across membrane
Why measure driving forces?
- quantitative approach will exorcise mistaken idea that ATP magical ‘high energy compound’
- can calc conditions req for eq and how far a reaction is displaced from eq
- displacement defines capacity of reaction to perform useful work
How does Gibbs energy and entropy vary in diff systems?
- isolated system = no exchange of energy and materials
- closed system = ΔH, heat flows
- open system = living systems exchange energy w/ surroundings
What is the Gibbs equation?
- ΔG = ΔH - TΔS
What does a value of less than 0 for ΔH mean?
- net increase in entropy of system and surroundings
- ∴ spontaneous process
How can Gibbs energy constant be observed as a function of its displacement from eq?
- if push reaction further away from eq, Gibbs energy changes
- if put energy in poss to move reaction left/right and increase Gibbs
Does the eq constant (K) have units?
- yes unless no. product molecules = no. substrate molecules
What relation must K and T (temp constant) have in order for ΔG to be +ve/-ve?
- ΔG -ve if TK
How can ΔG be calc using mass action ratio?
- ΔG = -2.3RT log10 [K/Γ)
- R = gas constant
- T = temp constant
- Γ = observed mass action ratio
What does the equation ΔG = -2.3RT log10 [K/Γ) tell us about ΔG?
- has value that is function of displacement from eq
- means 37° reaction maintained 1 order of magnitude from eq has ΔG of 5.9kJ/mol
Are spontaneous/non spontaneous processes reversible?
- spontaneous = only reversible w/ difficulty
- non spontaneous = reversible
How does cell make reactions spontaneous in matrix and cyto?
- pushes reaction away from eq
What defines the capacity of reactants to do work?
- extent to which observed Γ displaced from eq
Why is the high energy phosphate bond a myth?
- hypothetical cell could utilise any reaction to transduce energy from mito, if maintained certain no. orders from eq
- but is important that eq constant for ATP hydrolysis has approx value it does, as provides sufficient driving force for many +ΔG processes
What is membrane pot?
- diff in electrical pot between 2 aq compartments separated by membrane
How is ΔpH defined?
- pH in P phase - pH in N phase
In respiring mito is ΔpH usually +ve or -ve?
- -ve
How is Δψ defined, and is it usually +ve or -ve?
- P-phase - N-phase
- usually +ve
What can Δp be made up of?
- ΔpH and Δcharge (more H+ on 1 side)
- Δcharge only (more +ve ions on 1 side, same no. H+)
- ΔpH only (same no +ve ions, more H+ on 1 side)
What is DNP, and why is it dangerous?
- an uncoupling agent (protonophores)
- allows H+ in mito to link back to other side w/o doing useful work
What is a protonophore?
- proton carrier
What is an ionophore?
- carriers of ions across membrane
When are ATP, ADP and inorganic phosphate no use?
- when at equilibrium
What is the structure of ATPase?
- F1 is catalytic part –> formed by α3 β3 hexamer w/ γ subunit inside it, and ε attached to γ
- subunit δ bound to ‘top’ of hexamer and subunits b
- hydrophobic transmembrane segment of b in contact w/ a
- γ and ε bound to ring shaped oligomer of c-subunits
- proton translocation takes place at interface of subunits a and c
What is the catalytic mechanism of ATPase?
- rotary catalysis
- involves rotation of γ subunit w/ ε and c subunit oligomer relative to rest of enzyme
What is the binding change mechanism for ATP synthesis?
- DIAGRAM*
- central asymmetric cam rotates in step 1 relative to 3 α/β subunit pairs
- forces 3 catalytic sites to undergo conformational changes
- tight = where ATP made
- loose = where substrates come in and bind
- open = substrates ADP+Pi can easily come in
How is ATP made?
- pmf drives protons through –> drives rotation of c-subunit oligomer ring relative to α and β subunits
- rotation passed to γ and ε bound to c-subunit oligomer ring
- rotation of asymmetric γ mechanistically causes conformational changes in αβ hexamer, each 120°γ rotation forces 1 of 3 catalytic sites located at αβ interface into open conformation
- freshly synthesised ATP released and ADP+Pi bound instead, high affinity of opened site to phosphate impairs ATP rebinding and favours ADP binding
- rotation cont, forcing next site into open conformation, ADP+Pi bound to previous O site occluded and ATP synthesis occurs
- ATP formed released when γ makes 1 360° turn and once again open site
What have more recent experiments revealed about how ATP is made?
- that if γ mechanistically forced into rotation, ATP synthesis takes place even w/o proton translocating F0 part
How many protons req to make ATP?
- complete 360° req passage of no. of protons = no. of subunits
- generally agreed that 1 rotation of γ w/in αβ synthesises 3ATP
- so H+/ATP might be 3.3 (ie. 3H+ needed to make 1ATP
What does no H+ req to make ATP depend upon?
- no. c-subunits
What input of Gibbs energy is req to maintain [ATP]/[ADP] ratio?
- 57kJ/mol to keep ratio 10^10 away from eq
What are the muscles of inspiration?
- accessory = sternocleidomastoid, scalenes group, pectoralis major
- principal = external intercostals, diaphragm
What are the muscles of expiration?
- quiet breathing = passive elastic recoil of lungs, rib cage and diaphragm
Is it poss to reverse molecular respiration?
- yes, if put energy into system
* DIAGRAM*
What is cytochrome c?
- soluble e- carrier in mito/bacteria etc.
- most widely used e- transporters
What is the structure of cytochrome c?
- haem in centre
- square planar structure w/ central Fe, coord by 4 pyrrole rings
- Fe cycles between Fe(II), Fe(III) and occasionally Fe(IV)
- same prosthetic group in Hb and myoglobin
What is ubiquinone?
- lipid soluble carrier of H
What is the e- tower of redox pots?
- in general e-s flow from -ve to +ve redox pot
What is the redox reaction of cytochrome c?
- cyt c [Fe(III)] + e- cyt c [Fe(II)]
What is the redox reaction of ubiquinone (UQ)?
- UQ + 2e- + 2H+ UQH2
What is the biggest redox pot in most mito and bacteria?
- NAD+/NADH to 1/2O2/H2O
What are the diff redox carriers (complexes) in mito chain?
- complex I = NADH deHase, FMN, 8 Fe/S centres
- complex II = succinate deHase, FAD, 3Fe/S, haem b
- complex III = cytochrome bc, complex II haem b, Fe/S, c1
- complex IV = 2x haem a, 3 Cu
What is the pathway of redox carriers in mito chain?
- complex I, glycerol-3-P-deHase and complex II feed into UQ/UQ2 pool
- feeds to complex III
- then to complex IV
Does H+ pumping in mito chain exceed H+/ATP ratio of 3?
- yes, greatly exceeds it
Do e- carriers all ox or red?
- rarely, grad of e- occupancy
Unlike mito resp chain, resp apparatus of bacteria is what?
- highly flexible
How is the resp apparatus of bacteria flexible?
- modular –> mix and match assembly of components put together to achieve particular function
- numerous deHases, several types of quinones, 3 oxidases and numerous anaerobic reductases
- so system must be branched
- composition and ∴ function is controlled by circuits of gene reg in response to growth conditions
Is NADH deHase of E. Coli a proton pump?
- yes
What is the 1st terminal oxidase of E. Coli?
- cytochrome bo’ or Cyo, a proton pump
- H+/e- = 2
What is the result of coupling NADH deHase to cytochrome bo’?
- makes efficient proton pumping resp chain
What are the 2nd and 3rd oxidases of E. Coli, and what is there role?
- NOT pumps, but DO translocate protons
- structurally and functionally similar
- 2nd is CydAB
- 3rd is AppBC or bd-II
- spatially separate e- and H+ from ubiquinol
- H+/e- = 1
How can energy conservation be manipulated?
- expression of diff deHases and oxidases
- routes reg at level of gene expression
What are the characteristics of Azotobacter vinelandii?
- obligate aerobe
- carries out N fixation which is v O sensitive
- has 1 of highest resp capacities known
- removes internal O by phenomenal oxidase activity of cytochrome bd
- bd mutant cannot grow by N fixation in air
What are the energetics of growth at pH 1-3?
- favourable pH grad
- balanced by internal +ve membrane pot due to K+
- to help prevent massive proton influx a membrane pot is used
What are energetics of an alkaliphile based on N+ circulation?
- resp chains pump Na+ not H+
- decarboxylase pumps Na+ outwards to contribute to Na grad
- ATP synthase and flagellar rotation driven by Na+ flux
- antiporter used to exchange Na+ for H+
How does pmf have a central role in bacteria w/ diff lifestyles?
- resp and glycolysis in facultative like E. Coli
- fermentative metabolism only, in bacteria like streptococci and clostridia
- photolithotrophic metabolism in green and purple sulphur bacteria
