Electron Transport Chains and Photosynthesis Flashcards
give an overview of the production of energy from glucose?
- oxidation
- high energy electrons carried by NADH
- electron transport chain
- ATP produced
- oxygen is the final electron acceptor
how much energy is produced by glucose?
free energy = -2850kJ/mol
energy density of 17mJ/Kg
how much NADH is produced at each stage?
2 mol of NADH produced in glycolysis (cytosolic)
2 mol of NADH from oxidation of pyruvate (mitochondrial)
6 mol of NADH in the citric acid cycle (mitochondrial)
describe the electron transport chain
- 4 big complexes
- ATP synthase
- accepts electrons
- travel through different components
- oxygen = final acceptor
- bacteria can use other molecules
- energy produced is used to pump protons
what is the purpose of the proton pump?
- pumps protons from the matrix into the intermembrane space
- forms a proton gradient
- used to energize the ATP synthase
describe the reduction of NAD+ to NADH
- endergonic
- happens on nicotinamide
- involves 2 electrons and proton
- redox potential: E0 = -0.32V
what does a negative redox potential mean?
- reaction is not spontaneous
- doesn’t want to move in that direction
what happens when NADH feeds electrons into the electron transport chain?
- oxidised to NAD+
- oxygen accepts electrons and is reduced to water
- coupled to formation of ATP
what percentage of our energy is conserved?
70-80%
what is the pathway from NADH to O2?
- not a single step reaction
- multiple steps allow conservation of free energy (as ATP) at discrete points in the pathway
- this reaction sequence is accomplished by the respiratory chain which is an e- transport pathway
- the enzymes that catalyse the reactions of the repiratory chain are all membrane bound
what are the 4 main complexes
- It goes Complex 1 → Complex 3 → Complex 4
- Complex 2 feeds in from the citric pathway between complex 1 and complex 3
what happens at complex I?
- NADH → UQ Oxidoreductase
- Catalyzes transfer of 2e- from NADH to ubiquinone
- NADH oxidised initially by a flavoprotein, contains flavin mononucleotide (FMN) as a prosthetic group
- Electrons then passed to Fe/S centres
- each centre is reduced by just 1e
- Energy produced is conserved in conformational changes
- When electrons hop through the chain of Fe/S clusters every time a little bit of energy is used to cause a conformational change → attached to a part of the protein which pushes protons from one end to the other end
what is the structure of complex I?
- Fe/S centres = iron sulfur centre (non- haem iron proteins) → each Fe/S centre is covalently linked to Cys residues in protein
- Arrangement of FeS clusters forms the ‘electron wire’
Multiprotein complex
what is complex I inhibited by?
rotenone/NO
what happens at complex II?
essentially a part of the citric acid cycle
Succinate dehydrogenase
Catalyses oxidation of succinate and reduction of UQ
1 flavin adenine dinucleotide (FAD which is reduced to FADH2 covalently bound to protein) → delivers electrons
3 Fe/S centres
what happens at complex III?
- UQ-Cyt c oxidoreductase
- Each cytochrome is reduced by 1e-
- The Fe atom coordinated in the porphyrin ring is reduced: Fe3+ → Fe2+
what is the structure of complex III?
2 b-type cytochromes → named after their absorption of light
1 Fe/S centre (Rieske protein)
Cytochrome C1
Cytochromes: a haem prosthetic group bound to a protein
Iron is in its haem form → similar to haemoglobin
what is cytochrome C?
The only component of the respiratory chain which is not an integral part of the membrane
Nevertheless, it is bound loosely to the outer side of the inner membrane
Shuttles between complex III and complex IV → picks up electrons from 3 and moves them onto 4
what happens at complex IV?
- Cytochrome c oxidase
- Accepts 4e- from cytochrome c (=4 separate turnovers of cytochrome c)
- System essentially has to store electrons until there a 4
- When fully reduced can the reduce O2 together with 4H+ → 2H2O
- Oxygen reduction on matrix side of the membrane
what is the inhibtor of complex III?
antimycin
what is the structure of complex IV?
Cytochromes a1, a3
2 copper atoms (CuA, CuB) → easily reduced and oxidised
Multiprotein complex
what are the inhibitors of complex IV?
CN cyanide (blocks the copper centres for picking up electrons), CO (replaces oxygen on the binding site), azides
how can we determine which complexes actually make ATP?
- coupling ratios
- Can use oxygen electrodes to study the system
Measure O2 consumption by mitochondria in a closed chamber with O2 electrode and observe
Background respiration observed with respiratory substrate (NADH)
Addition of ADP → large increase in respiratory rate. When ADP is all phosphorylate, rate returns to background rate
i.e. the e- transport and phosphorylation reactions are COUPLED
If a known amount of ADP is added the amount of O2 used during phosphorylation can be measure to give:
ADP: O2 ration (P:O ratio)
P:O ratio is a measure of the number of ATP molecules synthesized per pair of e- passing down the respiratory chain
You can then look at different inhibitors and use different substrate
how can P:O ratios be used to identify the coupling sites in the respiratory chain?
Establish P:O with different substrate that act on different complexes
At which points of respiration chain is the thermodynamically downhill flow of electrons coupled to synthesis of ATP??
Conclude → complexes I, III, IV are all coupling sites but no ATP is produced by complex II
how can we use physical separation to study mitochondrail complexes?
Weak detergents disrupt lipid interactions
Purification of macromolecular components (protein complexes) catalysing specific partial reactions in e- transport pathway
Reconstitute on lipid vesicles
how is phosphorylation energized?
indirectly via generation of a H+ potential across the inner mitochondrial membrane
what is the chemiosmotic hypothesis?
- The coupling complex in the redox chain are H+ pumps
- Low membrane permeability to H+ allows build up of transmembrane H+ potential
- Passive return of H+ across the membrane energises ATP synthesis
what allows the chemiosmotic hypothesis to work?
- lipid membrane stops charged molecules
- Redox enzymes + ATP synthase are discrete element in the membrane
- Free energy is stored in the proton gradient → released to make ATP
what is the hydraulic analogy of chemiosmotic coupling?
Free energy input → e- transport Pump H+ pump Coupling energy: potential energy difference → have a high potential and a low potential Energy transducer → ATP synthase ATP
what is the F-type ATP synthase?
Two sectors → F0 and F1
Rotation → Sits in the membrane and turns around
ADP binding site → fixed → ATP formation
Turn around motion used to create ATP → runs off the ‘electric current’ created by protons
Complicated → lots of subunits
Catalytic part → sticks in the matrix
what is the chemical potential of an uncharged solute?
Chemical potential of S = μs = μos + RT ln [S]
Chemical potential difference (J/mol), inside with respect to outside is
ΔμS = (μS)i – (μS)o = RT ln { [S]i / [S]o }
- chemical potential is a function of the potential gradient
what is the electrochemical potential of a charged solute?
Electrochemical potential of Sz = μs = μos +RT ln[Sz] + zF
Electrochemical potential difference, inside with respect to outside is
ΔμS = (μS)i – (μS)o = RT ln { [Sz]i / [Sz]o } + zF
how do we write the electrochemical potential equation with protons?
can convert to volts → divide by F
proton motive force (PMF) = ΔμH/F
PMF = 60 (pHo – pHi) + AY
whhat does the electrochemical potential depend on?
concentration gradient and the membrane potential
A measure of the free energy in the electrochemical potential difference for protons across a membrane
describe PMF in coupled mitochondria
Inside of the matrix negative
Proton flows, stoichiometries and energetics
Every NADH → 2e-
I, III, IV = 4H, 4H, 2H
Use about 3 protons to make ATP → region of 33 ATPs per glucose
Total of 10H+ pumped out per 2e- flowing through redox chain
how many protons are need to make ATP?
- 3
- Energy available for ATP synthesis is:
But ΔG’ATP = -48.8 kJ/mol
i.e. for ATP synthesis, +48.8 kJ/mol is required
Reaction is nH+o + ADP + Pi ↔ nH+i + ATP
Energetically this reduces to nF(PMF) + ΔG’ATP < 0 for ATP generation
By coupling ATP synthesis to translocation for 3H+/ATP is available for synthesis of each mol of ATP
More than enough energy
Lose some energy where → energy is wasted in the form of heat → conditions where you actually might need some more
what is H+ flows and what is the impact on redox-phosphorylation coupling?
- ADP facilitates flow of H+ through ATP synthase
- Decreases the PMF slightly
- Decrease in opposing driving force enables faster operation of redox coupled H+ pumps
what is the action of uncouplers?
- Some compounds (Abolish ATP synthesis ‘uncouple’, Speeds up respiration)
- Becomes easy to pump because there is no gradient
- Make the membrane leaky
- Rapid speeding up
- Uncouples catalyse passive H+ flow (they are protonophores) and rapidly abolish PMF
- There’s no longer a driving force for ATP synthesis
- No forcing opposing redox reaction reactions
- Specific uncoupler proteins → particularly prevalent in adipose brown fat tissue
why can sepcific uncoupler proteins be useful?
hibernating animals
Uncouple mitochondria and use the system to generate heat
how is membrane bound ATP synthase is ubiquitous at energy coupling membranes?
Conversion of redox energy into chemical energy
Electrons → proton gradient
Same in many bacteria
Mitochondria + aerobic bacteria + anaerobic respiration → can use different molecules as the final acceptor
Fermenting bacteria → ATP synthase works in reverse, no respiration, pumps: ATPase, evolved to get rid of excess protons
Thylakoids: pumps and ATP synthase inverted compared with mitochondria, pumped into the lumen
what happens to free energy stored in the pump?
transduced into solute gradient energy by the carriers
describe PMF in flagellum?
Swimming → some bacteria swim towards attractants (chemotaxis)
Flagellum rotates
Rotation is powered by PMF
what are the energetics of photosynthesis?
Basic equation: CO2 + H2HA (light) → (CH2O) + H2O + 2A
A can be S but is usually O
Have an electron donor and an electron acceptor
what are the 4 separate categories of the photosynthesis equation?
- Absorption of light
- e- transport
- photo(phosphorylation)
- CO2 fixation
where do reactions 1-3 of photosynthesis occur?
thylakoid membrane
what are the different parts of the chloroplast?
Thylakoid Envelope (double membrane) Thylakoid lumen Stroma Granal sack Stromal lamella
what is light?
- electromagnetic radiation
- visible light is at 370 - 750 nm
- C = f * y (speed of the wave)
- blue, green and red light are the main ones
what is a photon?
particle carrying a quantum of light
what is the energy of a photon?
Energy of a photon is inversely proportional to wavelength or proportional to its frequency
E = hc/λ
E = hf
h = Planck’s constant
c = speed of light
Like any other molecular particle we can speak of Avogadro’s Number (or mole of photons) → 1 mole of photons = 6 x10^23
what are the primary photosynthetic pighment in higher plants?
- chlorophyll a and chlorophyll b
- very hydrophobic molecules localised exclusively within the membrane and flavonoids and carotenoids
- attached to side chains and cofactor
- absorbs energy → by exciting electrons in its orbital
describe the absorption of chl a and b?
Absorption of blue photon pushes e- to 2nd excited state
Only sufficient energy in red photon to get to 1st state
E- concerned is delocalised over tetrapyrrole ring by alternating single and double bands
Transitions 2nd → 1st excited state: energy lost as heat
1st excited state → ground state: energy can be lost through emission of photon of longer wavelength = fluorescence
Can actually measure the fluorescence → can use it as a diagnostic
what is the 3rd form of energy loss that are needed to accomplish light harvesting?
resonance energy transfer
what is resonance energy transfer?
- Neighbouring chl responds to electric field of excited chl
- e- of chl excites a lower energy e- in neighbouring chl therebby losing its own energy
- There is a small energy loss in this process (almost 100% efficient)
- In chl aggregates excitation tends to pass from species absorbing at shorter wavelengths to those absorbing at longer wavelengths of light
what is the biological importance of resonance energy transfer?
- Proteins broad absorption spectrum
- Other pigments absorb in other parts of spectrum are present
- Energy is funnelled to chls absorbing lower energy photons
- This process is known as light harvesting
- The chlorophylls are often bound to proteins which modifies them
- Resonance transfer allows the photon energy to be transferred to the reaction centre
what is light harvesting?
- Reaction centre
- Light harvesting complexes → all funnel to one reaction centre
- Broad spectrum of wavelengths of visible light is capable of funnelling energy to chl absorbing at long wavelengths
energy passed all the way to a reaction centre - Clusters → light harvesting complex → consists of chlorophyll and proteins
what is the antenna and reaction centre chlorophylls?
The Bulk of chlorophyll is in the light harvesting complexes
1 protein typically binds:
- Each reaction centre is associated with 300 antenna chlorophyll molecules
- Each reaction centre has its own special chlorophyll for participation in redox reactions
what are the 2 reaction centres in cholorplasts?
Photosystem I and II
what is photosystem I?
- absorption properties are different
- absorbs at 700 nm pigment is p700
- found in the stromal lamella
what is photosystem II?
- absorbs at 680 nm pigment is p680
- found in the granal lamellae
what happens at the reaction centre?
Energy arrives and is used in a redox reaction: the excited e- is lost from the chlorophyll to some other molecule
Resonance energy is converted into redox energy
The redox reaction is facilitated by a change in E0 of chlorophyll in the excited state
Energy is used to knock an election out of the chlorophyll can occur due to ring structures → energy can be delocalised
what happens in the redox reaction at the reaction centre?
Energy arrives in the form of resonance energy
Excites the P680
Electron leaves the chlorophylls
P680 becomes charged
Leads to the delivery of a new electron from water
what happens when the redox reaction is positive?
doesn’t want to lose an electron → highly oxidising (eg +1100)
what happens when the redox reaction is negative?
keen to lose an electron → highly reducing (eg -650)
what is the efficiency of the photosystems?
PSII has a 96% efficiency
PSII has a 88% efficiency
In both PSI and PSII there is a dramatic increase in propensity to donate e-
describe the initial redox reactions in PS II and PS I?
- transmembrane events
- build up a charge across the membrane as well → high voltage → pigments can respond to the voltage
what is the series arrangement of PSII and PSI?
Long y light: Oxidation of redox components between PSII and PSI
Shorter y light: Reduction of redox components between PSII and PSI
P680 pushing electrons out and P700 pulling them in
PSII and PSI act in series to catalyse electron flow between H20 and NADP+
The whole process is the reverse of what we see in the mitochondria → put energy in → needs energy to run
how does PSII and PSI act in a series to catalyse electron flow between H2O and NADP+?
- Water acts as an electron donor
- Enter photosystem II
- Go through a number of components
- Go through PSI
- Results in NADPH
- Use light → resonance energy transfer
- Changed from a strong oxidant to a strong reductant
what is used to pump protons?
free energy
what is the oxygen (evolving complex) redox component?
- Associated with PSII → 3 proteins
- On the luminal side of the membrane
- Active centre: 4 tightly bound Mn2+ ions
- Catalysed reaction: 2H2O → O
- The e- electrons are passed one at a time via tyrosine residues to oxidized P680+ reaction centres (potential source of ROS)
what is the PSII reaction centre redox component?
- Compromises a supramolecular complex
- Several distinct proteins binding redox chain components
- P680: chl a dimer
- Pheophytin (chl without Mg2+)
- 2 molecules of plastoquinone bound to specific proteins: PQa (tight) PQb (loose → diffuses within the membrane)
what is the plastoquinone redox component?
- Reduction of PQb is prevents by a number of herbicides → once reduced to plastoquinol the Qb molecules diffuses into the PQ pool
- The PQ pool: a large number of molecules of PQ freely dissolved in the hydrophobic portion of the the thylakoid membrane
what is the cytochrome b6f complex redox component?
- A supramolecular complex accepting e- form from PQ
- Comprises of: 2 spectroscopically distinct by type cytochromes, Cytochrome f, An fe2s2 centre, Binds PQH2
- Part of the system that pumps protons → pumps into the lumen of the thylakoid
how is the cytochrome b6f complex structurally and functionally similar to complex III in mitochondria?
- Both inhibited by Antimycin A
- Both accept a e- from quinol
- Structurally similar
- The b type cytochrome (2 haem groups bound to a single apoprotein) → sequence homology to mitochondria
- Both contain a “high potential” (E’0 = + 300 mV) Fe2S2 centre
- All these factors point to a common evolutionary origin of complex III and cytochrome b6f complex
what is lateral heterogeneity and plastoquinone diffusion in the cytochrome b6f complex?
Quite a long distance apart
Cyt b6f complex and PSI are in stromal lamellae
PSII is in the granal sack
Plastoquinone is a very mobile molecule which diffuses in the plane of the molecules → plastoquinone gets reduced and sits in the membrane and activates a kinase
what is plastocyanin?
small water soluble copper containing protein locked in thylakoid lumen
what is the PSI reaction centre?
Oxidises PC → 3rd supramolecular complex compromising: P700 Chl 6 additional Chls 2 quinones 3 Fe4S4 centres All help move electrons across membrane to next component 17 subunits and 6 chlorophylls Form 2 electron “wires” across membrane
what is ferredoxin?
A small protein with an Fe2S2 centre
Loosely associated with the stromal side of the thylakoid membrane
what is ferredoxin - NADP oxidoreductase (FNR)?
A flavoprotein containing FAD
Also located on the stromal side of the thylakoid membrane
Picks up 2 e- and a H+ to reduced NADPH
which of the steps are sensitive to specific inhibitors in photosynthesis?
Reduction of Qb by DCMU and atrazine
Reduction of cyt f by antimycin
Reduction of Fd by paraquat
what are the useful products of photosynthesis?
- NADPH → subsequently used in reduction of CO2 (‘dark reactions’)
- PMF → e- transport chain pumps H+ into the lumen hence ATP is synthesised (4 protons to make ATP)
what is the magnitude of PMF?
PMF is inverted compared with mitochondria
Orientation of ATP synthases is inverted → ATP made on outside of thylakoid membrane, in stroma
Stoichiometries: for redox chain, 6H+/2e-. For ATP synthase, 4H+/ATP
For each pair of e- passing through chain: 1 NADPH and 1.6 ATP are produced
what is cyclic e- transport and variable ATP/NADPH production?
- Observation → light of wavelength >680 nm results in a PMF but no net production of reducing equivalents
- Interpretation → the electrons cycle around through this system, still goes through b6f complex which pumps protons
PSI is excited by long wavelength light → electrons recycle through ferredoxin b6f complex and plastocyanin
Net production of NADPH is not possible because there is no reductant available but ATP can be produced
Cyclic electron transport might provide plants with a way of producing ATP if demand is high
why is efficiency of photosynthesis around 40%?
- not all light is used
- too much light results in radicals and UV damage
what is carbon fixation?
- ribulose and RuBisCO
- does not need direct light
- takes place in the stroma
- CO2 + ribulose 1,5 bisphoshpate → 2 3-phosphoglycerate phosphate
- exergonic
what is RuBisCO?
- most abundant protein
- Very low turnover rate
- Reaction energetics → -52 → spontaneous
- large allosteric enzyme
- 8 large and 8 small subunits
- mg2+ cofactor
how does mg2+ work in RuBisCO?
- released from thylakoid lumen in exchange for H+ during electron transport activates RuBisCO in stroma
- form of control/regulation
why is RuBisCO so slow?
- originated from around 2 billion years ago when CO2 concentrations were much higher
- much more substrate so the enzyme would have run faster
how are hexoses assimilated?
2 3-phosphoglycerate → need to energise by adding another phosphate group → catalysed by a kinase
Gets reduced by a dehydrogenase enzyme
Glyceraldehyde 3 phosphate isomer of dihydroxyacetone phosphate and aldolase catalyses a condensation reaction to get fructose 1-6 bisphosphate
Takes off a phosphate to get fructose 6 phosphate
describe assimilation of carbon in plants (also known as the ‘dark’ reactions)
- CO2 fixation → rubisco
CO2 + Ribulose 1,5–bisphosphate 2 (3-phosphoglycerate) - Reduction → cf gluconeogenesis
2 (3- Phosphoglycerate) → Fructose 6-Phosphate
how is ribulose 1,5 bisphosphate regenerated?
- making a 5C sugar from 6C and 3C sugars
- enzymes: transketolase and aldoalse
- cycle driven by reactions with large -ve delta G
RuBisCO
Fructose 1,6 bisphosphate
Phospho Ribulose kinase
what does transketolase do?
transfers 2C unit from a ketose to an aldose
what does aldolase?
condensation between DHAP and an aldehyde
outline the calvin cycle
RuBisCO → fixation of carbon
Energisation and reduction
Get 12 glyceraldehyde 3-P
Then get 6 G3P
Take 2 G3P → aldolase → fructose 1,6 bp → phosphatase
Use a ketolase → can move a 2C3 → 2x5 then enter the rest of the system
Get 2 C4 compounds can be fused to another 2 C3 compounds → 2 C7 compounds → etc
We end up with 6 ribulose molecules after another energisation step
what are the 3 major phases of the calvin cycle?
- carboxylation →
- reduction
- regeneration
what are many of the enzymes involved in photosynthesis activated by?
light
what are the energetics of CO2 assimilation to fructose 6 phosphate from 6 CO2?
- 1 ATP consumed in phosphorylating each mole of 3 PGA
- Since 6 CO2 + 6 Ru 1,5 BP → 12 PGA
- 12 ATP consumed here
- 1 ATP consumed in regenerating each mole of Ru 1,5 BP in the Calvin cycle
- Since 6 Ru 1,5 BP required for fixation 6CO2
- 6 ATP consumed here
- Overall, 18 ATP consumed - 1 NADPH consumed in reducing each mole of 1,3 BPG
12 NADPH consumed here
what are the fates of fixed carbon?
starch or sucrose
what is starch?
- important in diet
- food = bread, pasta
- storage polysaccharide
where does starch synthesis occur?
chloroplast of stroma
when is starch synthesis favoured?
when CO2 fixation rate exceeds the utilisation rate of reduced C
What are the 2 key reactions of starch synthesis?
ADP-glucose Pyrophosphorylase Glu 1-P + ATP → ADP-Gluc + PPi Starch synthase ADP-Glu + glucose → glucose + ADP
when is starch consumed and made?
made = during the day consumed = at night
where does sucrose synthesis occur?
in the cytosol
describe sucrose: export of reduced CO2 from the stroma?
- Reduced carbon comes out of the chloroplast and many transporters in the chloroplast membrane
- Exchanger at inner envelope membrane ensures that P exported as triose - P is replenished
describe sucorse: synthesis from trioses in cytosol
Sucrose: the major mobilisable sugar in plants
Moves from leaves the developing tissues such as roots as respiratory metabolite
What is photrespiration?
Rubisco fixes O2 as well as CO2
This is the oxygenase function of Rubisco
The reaction is unwanted → an evolutionary consequence of O2 in the atmosphere
For every 2 or 3 carboxylates you get an oxygenation
O2 competes with CO2 at the active site
what is the result of O2 + Ru 1,5 BP?
get phosphoglycolate
what 3 organelles does the recovery of C skeletons during phot respiration require?
Chloroplast
Mitochondria
Peroxisome
how is the loss of carbon dealt with?
Get a phosphoglycolate → undergoes conversion to glycine
Glycine moves into the mitochondria
2 glycines = 4 carbon, one of the carbons is split of into CO2
NH3 goes off → ended up with a carbon 3 serine
Undergo several changes to form glycerate and then 3 phosphoglycerate
This can the enter the calvin cycle
3 rescued and one is lost → ¾ of C is recovered
what is meant by C4 and CAM pathways?
Spatial and temporal CO2 concentrating mechanism to reduce O2 competition for Rubisco
The problem: as temperature increases O2 fixing ability of Rubisco increases more steeply than CO2 fixing ability
The solution: devise a mechanism for local enrichment at the site of CO2 fixation (Co2 pumps)
where is the C4 pathway found?
observed in tropical species eg sugar can, maize
what enzyme does C4 metabolism use?
PEP carboxylase
what is C4 metabolism?
Carries out a carboxylation
Picks up the CO2 in the form of bicarbonate
Makes malate → C4 sugar
Accumulate lots of malate in storage parts of the cell
Moves from the mesophyll into the bundle sheath cell and is split to release CO2 → enters the normal calvin cycle
There is a cost → 12 ATP extra per C6
What is CAM metabolism?
but temporal separation
Plants that live in the desert → have a risk of drying out when getting CO2
They open stomata during the night and capture carbon → store as malate
Close the stomata during the day and proceed with the Calvin cycle
