Oxidative Phosphorylation Flashcards
Name two oxidative processes ?
- Catabolism of glucose to pyruvate in glycolysis
2. Breakdown of pyruvate to CO2 and water in citric acid cycle
Where are the enzymes from carbohydrate oxidation transferred to and what do they form?
- NAD+ and FAD
2. NADH and FADH2
What are fatty acids and amino acids catabolised by and what do they use ?
- Oxidative pathways
2. NAD+ and FAD
What does the continued availability of NAD+ and FAD as electron acceptors depend on ?
The reoxidation of NADH and FADH2 by 02
What is oxidative phosphorylation ?
The process by which the free energy derived from glucose and other substrates and stored in NADH and FADH2 is used to generate ATP from ADP and an inorganic phosphate
Where is the major site of oxidative phosphorylation in mammalian cells ?
The mitochondria
How does the transfer of electrons from reduced coenzymes to O2 occur ?
By a stepwise flow of electrons through a chain of intermediate electron carriers
Where is the stepwise flow of intermediate electron carriers situated ?
The mitochondrial inner membrane
What do the electrons pass along ?
The electron transport chain to the cytochrome oxidase
What is the cytochrome oxidase also known as ?
Complex IV
What is the final electron acceptor ?
O2
What happens at the final electron accepter ?
O2 is reduced to water
What happens to protons as electrons pass along the electron transport chain ?
Protons are translocated from the mitochondrial matrix across the inner membrane into the intermembrane space
What does the translocation of protons set up ?
A proton electrochemical gradient across the mitochondrial inner membrane
What does delta p stand for ?
Proton electrochemical gradient
What drives the synthesis of ATP from ADP and an inorganic phosphate ?
The flow of protons back across the membrane through ATP synthase
What is ATP synthase also known as ?
Complex V
What is ATP synthesis by mitochondria coupled to and by what ?
- Electron transport
2. Electrochemical gradient
What is ATP synthesis driven by ?
The free energy derived from the oxidation of substrates
In mammals, what is the majority of the oxygen that we consume a result of ?
ATP synthesis
What is the ATP produced used to do ?
- Maintain ion gradients
- Transport substrates and proteins across membranes
- Protein synthesis
- DNA synthesis
- Muscle contraction
What can electron flow and the resulting reduction of oxygen by suspensions of isolated mitochondria be demonstrated in a laboratory by ?
An oxygen electrode
What does an oxygen electrode consist of ?
A silver/silver chloride reference half cell joined to a platinum/O2 cathode by a saturated KCl bridge
What is the electrode compartment isolated from ?
The reaction chamber
What is a reaction chamber which is isolated from the electrode compartment ?
Where the mitochondria and their substrates or inhibitors interact
What isolated the electrode compartment from the reaction chamber ?
A thin teflon membrane
What is a teflon membrane ?
A membrane which is permeable to oxygen and allows oxygen to reach the cathode
What happens once oxygen reaches the cathode ?
It is electrochemically reduced
What does the reduction of oxygen at the cathode allow ?
A current to flow
What is mitochondrial electron transport monitored by ?
Measuring the rate of oxygen consumption
What is an example of an oxygen donor/oxidisable substrate ?
Succinate
What does stirring result in ?
Ensures the mixture is fully oxygenated
What does the blank rate represent ?
Respiration and ATP synthesis using substrates and ADP that were present on the mitochondria when they were isolated
At what point in the electron transport chain do electrons from succinate enter ?
Complex II
What happens upon addition of succinate and why ?
The rate of oxygen uptake increases as electrons from the added succinate move down the electron transport chain to O2
What happens after ADP is added to the reaction chamber and why ?
A sudden burst of oxygen uptake as ADP is converted into ATP
What is the actively respiring state called ?
State 3 respiration
Why does the trace curve upwards after about 2.5 minutes ?
The rate of oxygen consumption is slowing down as oxygen becomes limiting
What is electron transport limited by ?
ATP synthesis
What is electron transport coupled to ?
ATP synthesis
What can electron transport also be known as ?
Respiration
What is the respiratory control ratio ?
State 3 rate : State 4 rate
What does the respiratory control ratio indicate ?
The tightness of the coupling between respiration and phosphorylation
Why might the coupling not be perfect ?
Mechanical damage to mitochondria during the isolation procedure
What does the further addition of ADP restore the rate to ?
State 3
Why is state 4 respiration usually faster than the original rate before the first addition of ADP (state 2)?
Some ATP is broken down by ATPase activities contaminating the preparation and the resulting ADP is then re-phosphorylated by the intact mitochondria
Why does oxygen consumption decrease when ADP becomes limiting?
the flow of protons through ATP synthase cannot occur because movement of protons through the complex into the matrix is obligatorily coupled to the synthesis of ATP
What happens to the proton electrochemical gradient in the absence of ADP ?
The gradient quickly reaches and is maintained at maximum value
What happens if the proton electrochemical gradient is already at a maximal value ?
The system is fully charged and further proton pumping cannot occur
What happens if the proton electrochemical gradient is at a maximum value and further proton pumping cannot occur ?
Proton electrochemical gradient applies an energetic back pressure preventing further proton pumping by the proton transport chain
What are chemical uncouplers ?
Molecules that can ferry protons from the inter membrane space across the mitochondrial inner membrane and into the matrix
What does an uncoupler facilitate ?
Protons moving back into the matrix without going through ATP synthase
What happens when protons are pumped without any back pressure from the gradient, ie protons are pumped out and returned via an uncoupler ?
Electron flow through the electron transport chain increases but the free energy available is not linked to production of ATP but is lost as heat
What is an example of an uncoupler ?
2,4-DNP
What happens upon the addition of 2,4-DNP and why ?
Respiration increases as there is no longer a limiting supply of ADP and state 4 is not achieved
What is work done on transporting electrons being lost as ?
Heat
What is malate ?
A source of NADH
What is rotenone ?
A poison/inhibitor
When does rotenone block electron transport ?
When NADH is the source of electrons but not when succinate is the source of electrons
Where do electrons from succinate enter the electron transport chain ?
Complex 2
What does rotenone block ?
Electron transport between complex I and complex II.
Which electrons can by pass the rotenone block ?
Electrons from succinate
Where does antimycin block and why ?
Complex III, succinate cannot pass the antimycin block therefore it must be beyond complex II
What is bongkrekic acid ?
An inhibitor of the translocator
What does atractyloside do ?
Limits ADP in matrix
What does oligomycin do ?
Binds to the F0 and inhibits F1F0ATPase- blocks proton channel
What is calcium uptake driven by ?
Membrane potential
What happens when calcium ions are added ?
Rate of oxygen consumption increases
Why does the rate of oxygen consumption increase on addition of calcium ?
Positive ions are taken uptake via the calcium ion uniporter driven by difference in membrane potential
What is ruthenium red ?
An inhibitor of the uniporter
What is the malate-aspartate shuttle ?
How NADH is transferred across the mitochondrial inner membrane by the shuttle involving glutamate and malate
What do redox centers contain ?
Metal ions such as iron, copper and manganese
How many cytochromes are there in the respiratory chain of eukaryotes ?
4
What are the cytochromes involved in the respiratory chain of eukaryotes ?
- aa3
- b
- c
- c1
Is cytochrome C part of a complex ?
No
What is cytochrome C since its not in a complex ?
Mobile
What are some ubiquinones ?
- Coenzyme Q10
What is an ubiquinone ?
A lipophilic mobile redox center
How many complexes is in the electron transport chain ?
4
What is a respirasome ?
A super complex
What enters complex I ?
Electrons from NADH
How do electrons get from complex I to complex III ?
Via a coenzyme Q
How do electrons move from complex III to complex IV ?
Via cytochrome C
What happens to electrons at complex IV ?
The electrons are transferred to oxygen which is reduced to water
Where do electrons from succinate enter the chain ?
Complex II
What do the electrons from the oxidation of succinate, which enter the chain at complex II do ?
Feed the electrons into complex III via CoQ
What is another name for complex I ?
NADH-ubiquinone oxidoreductase
What does complex I contain ?
Multiple electron carriers, flavin mononucleotide plus iron sulphur centers
What is the immediate electron acceptor from NADH in complex I
Flavin mononucleotide
What inhibits electron transfer in complex i ?
Rotenone
What is another name for complex II ?
Succinate-ubiquinone oxidoreductase
What is the inhibitor in complex II ?
Malonate - inhibits succinate oxidation
What inhibits electron transfer in complex III ?
Antimycin A
What is another name for complex IV ?
Cytochrome c oxidase
What inhibits electron transfer to O2 ?
Cyanide, CO and azide
How many electrons does complex IV accept ?
4
What is another name for ATP synthase ?
F1F0-ATPase
What is ATP synthase ?
Oligomer
What is the function of ATP synthase ?
ATP synthesis
What inhibits ATP synthase?
Oligomycin
What always generates a proton electrochemical gradient ?
Metabolic reactions that energise the membrane
What does energy coupling require ?
A topologically closed structure
What do uncouplers allow protons to do ?
To move down the electrochemical gradient, across the inner membrane back into the matrix without going through the ATP synthase
What are uncouplers ?
Lipophilic weak acids
