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189 Cards in this Set
- Front
- Back
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What do oxidation/reduction reactions involve?
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Transfer of electrons from a donor to an acceptor
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What is a reducing agent?
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Something that gives away an electron (donor)
Get oxidized |
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What is an oxidizing agent?
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Something that accepts an electron
Gets reduced |
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What is the principle use of oxygen?
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Respiration
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What is respiration?
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The controlled reaction by ETC of hydrogen and oxygen to form water + ATP
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What is ETC?
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The Electron Transport Chain- electrons are pased along protein complexes to lower energy states, they produce changes in the transport proteins, membrane, that leads to the pumping of protons across the IMM to the intermembrane space. These protons are stored for ATP synthesis
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How are the redox reations linked?
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In increasing ability to accept electrons
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Is the first component a good reducing agent?
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Yes- it gives up electrons readily, and therefore is a poor oxidizing agent
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What does it mean that as you progress down the ETC, you have a greater ability to accept electrons?
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There is increased oxidizing ability
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What is the first source of electrons in the ETC?
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NADH
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What is the ultimate electron acceptor in the ETC?
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O2
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What is a conjugate pair?
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Two species (redox couple) that can be interchanged through the loss or gain of electrons and possibly protons
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What is an example of a conjugate pair?
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Fe 3+ + e -> Fe2+
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Which component of this redox couple is the oxidizing agent/electron acceptor?
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Fe3+
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Which component of this electron couple is the reducing agent/electron donor?
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Fe2+
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Which unit is strandard reduction potential expressed in?
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Volts
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What does a half reaction show?
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Electrons being consumed to form the reduced part of the conjugate pair. This is done without showing the electron source
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What does standard reduction potential assess?
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It assesses the ability of a chemical species to be reduced or oxidized- depends on magnitude of value
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What type of property is the reduction potential of a component?
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It is an intrinsic property- meaning it cannot be deduced theoretically- must be found experimentally
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How can one experimentally determine the standard reduction potential?
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By an electrochemical cell
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What is the reference standard of the electrochemical cell?
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Hydrogen (H2) electrode
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What is the half-reaction at the H2 electrode?
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H+ + e-> 1/2 H2
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What is the standard reduction potential for the H+ electrode?
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O V at ph=0 (1M H+) and 1 atm pressure
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What type of metal is immersed in each cell?
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Platinum
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What is the purpose of the salt bridge?
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It maintains the electroneutrality by transfering ions. Because we are measuring the movement of electrons from one half cell to another, we would not want a charge differential to build up in half cells because this would effect the EMF. Ions from the salf bridge move into either half cell as required
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What happens if electrons flow from the half cell to the standard reference?
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Standard reduction potential would be negative, gives up electrons to H+ electrode
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What does a negative standard redution potential mean?
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Better reducing agents- negativity signifies a greater tendency to give up electrons
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What happens if electrons flow from the standard reference to the half cell?
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THe standard reduction potential is positive- signifies a better oxidizing agent (greater tendency to accept electrons)
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What is an example of a redox couple that has a negative standard reduction potential?
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NAD+ + H+ + 2e-> NADH
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What is an example of a redox couple that has a POSTIVE standard reduction potential?
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O2/H2O
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What is the change in standard reduction potential?
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Standard reduction potential (oxidizing agent) - Standard reduction potential (reducing agent)
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How would you determine if something can reduce another?
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Determine change in standard reduction potential and compute gibbs free energy change, and determine if the reaction is energetically favorable
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What is the relationship between gibbs free energy and standard reduction potential?
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Delta G standard= -nFdeltaEstandard
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What is n?
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The number of electrons being transferred
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What is F?
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Faraday constant: 96.5 kj/volt*mol
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How do you figure out if the reaction would occur in the cell?
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Compute non-standard change of Gibbs free energy
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What is the formula for the calculation of non-standard change in Gibbs free energy?
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DeltaG= DeltaGstandard + RT ln ([prod]/[reactant])
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What is the Nerst equation?
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Allos you to determine the reduction potential differences under NON STANDARD CONDITIONS
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Where are the components of the ETC located?
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The Inner Mitochondrial Membrane
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What are some features of the IMM?
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Many cristae (to increase surface area)
Rich in proteins (80% by weight) |
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What are reducing equivalents?
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Where the majority of energy released in oxidation of fatty acids, amino acids, carbohydrates is captured
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What are the reducing equivalents for the ETC? Where are they formed?
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NADH and FADH2 are reducing equivalents for the ETC, and they are formed in the TCA
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How is free energy generated in the ETC and what is it used for?
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Free energy is generated from converting high energy electrons to low energy electrons is used to pump H+ across the IMM to the intermembrane space
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How are the protons used to synthesize ATP?
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An electrochemical gradient of H+ is used to synthesize ATP.
H+ come back into the matrix, liberate free energy that gets used to make ATP |
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What is the only non-protein containing component in the ETC?
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CoQ (ubiquinone)
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What are the standard reduction potentials like in the ETC?
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The standard reduction potentials get more positive as you go down the ETC, they have a greater tendency to accept electrons, therefore having a lower reducing potential
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How many complexes are in the IMM?
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Four
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Where do electrons from NADH flow?
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To complex 1, then 3, 4
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Where do electrons from succinate flow?
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Complex 2, 3, 4
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How much energy is required for ATP synthesis?
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-30.5kJ/mol
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After passing through complex 1, where do the electrons go?
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To carrier CoQ
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Why do we only get 2 ATP when starting with succinate to complex 2, but 3 ATP when starting with NADH to complex 1?
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There is no free energy released by transferring the electrons from FADH2 to complex 2
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How is FADH2 produced?
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Oxidation of succinate to form fumarate by succinate dehydrogenase
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How would you calculate the efficiency of the electron transport from a reducing equivalent to oxygen?
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Calculate deltaGstandard.
Compute #ATP formed with that reducing equiv X 30.5 J / deltaG standard |
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How was the electron transfer sequence deduced?
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Using respiratory complex inhibitors that block e transport
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What blocks complex 1?
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Rotinone or amytol
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What blocks complex 3?
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Antimycin A
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What blocks complex 4?
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Cyanide (is a terminal inhibitor, combines with iron (Fe3+)
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What is betahydroxybutyrate?
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Source of electrons for NAD+
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What happens when succinate is added to a rotenone/amytal inhibited ETC?
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Allows bypass of block of ETC, can continue through complex 2
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What is TMPD/ascorbate?
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Allows bipass of complex 3 block by Antimycin A
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How is the reaction using different inhibitors performed?
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With an oxygen electrode measures the [O2], immersed in a mitochondrial suspension with an excess of ADP and Pi
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Why is the reaction performed in an excess of ADP and Pi?
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You do not want ETC to stall or slow down because of a lack of these- you want it to because a specific complex was blocked by an inhibitor
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What would a drop in [O2]in this experiment signify?
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Utilziation of O2 to produce H2O (by the ETC)
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What happens to ATP synthesis if you block ETC? Why?
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ATP synthesis stops because ETC and ATP synthesis are tightly coupled
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What happens to the ETC if you deplete ADP or Pi?
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ETC stops
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What is the P/O ratio?
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Number of ATP produced per 1/2 O2, or per NADH, or per FADH2
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Describe an experiment to calculate the P/O ratio
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Add a defined amount of ADP and unlimited amounts of Pi and e source. Therefore the limiting component is ADP.
Add ADP and e source and measure drop in O2. Once O2 stops dropping, you know all the ADP has been used up. We know that each mol of O2 handles 4e, and that eac NADH handles 2e. Therefore x concentration of O2 corresponds to 2x concentration of NADH. P/O ration= mol ADP/molNADH= #ATP/NADH |
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What if you block complex 1, and add succinate, what would your p/O ratio be?
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P/O ratio is 2
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What is the P/O ratio if complex 2 is also blocked?
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P/O ratio is 1
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What are redox active centers?
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Catalize reduction/oxidation, are associated with proteins
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What are the redox active centers in Complex 1?
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FMN, Fe-S
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What are the redox active centers in complex 3?
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Fe-S, cytochromes
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What are the redox active centerse in complex 4?
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Copper/Cytochrome
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Are redox active centers mobile? If not, how do the electrons transport?
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They move through the polypeptide chain backbone
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Which complex is the largest in the ETC?
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Complex 1, MW 850 000
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What type of proteins are the complexes in the ETC?
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Intergral- span membrane completely. Therefore, they can only be extracted by disrupting the membrane with a detergent
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What are iron sulfur clusters?
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Redox active proteins, can be [2Fe-2S] or [4Fe-4S]. Redox states differ by 1 formal charge, and therefore can only carry 1 electron at a time
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What are the redox active centers in cytochromes?
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Heme groups
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What does the heme contain?
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Porphyrin ring/central ring protion
Different substituents depending on heme type |
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How many electrons at a time do cytochromes transport?
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1e
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What are the two shuttles in the ETC?
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COQ (Coenzyme Q)
Cytochrome C |
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For which complexes does CoQ act as a shuttle?
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From complex 1 to 2
From complex 2 to 3 |
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For which complexes does Cytochrome C act as a shuttle?
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Complex 3 to 4
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What is special about the structure of CoQ?
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It has a long hydrophobic tail made of ISOPRENOID UNITS
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What is an isoprenoid unit?
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A 5C unit
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For mammalian CoQs, how many isoprenoid units does it have?
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n=10, therefore 10 units, (50C)
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Why are isoprenoid units present in the CoQ?
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To increase its hydrophobicity. The headgroup of CoQ is quite polar, and we need to increase its solubility in the membrane (lipid bilayer). Consequently, the long tail is essential if you want CoQ to diffuse within or across the lipid bilayer
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How does CoQ normally exist?
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In an oxidized state (quinone form- Ubiquinone)
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What happens if an H+ is added to the oxidized CoQ?
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You get the semiquinone form= Coenzyme QH (Ubisemiquinone)
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What type of molecule is Coenzyme QH?
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It is a free radical form
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What is a free radical?
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Damaging species generated in metabolism
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What happens if we add a second H+?
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Reduced or hydroquinone form is generated
-CoenzymeQH2 or ubiquinol |
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Consequently, how many electrons can CoQ carry?
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1 or 2
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What are some properties of cytochrome c?
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Its a small protein
Highly conserved phylogenetically Length 104-100 aa Mw 12000-13000 Coloured (reddish brown) |
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What does cytochrome c contain?
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1 heme group that fluctuates between two oxidation states (Fe2+, Fe3+)
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How many electrons can cytochrome c carry?
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1 at a time
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What type of protein is cytochrome c?
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Peripheral membrane protein?
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Where is cytochrome c associated?
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With the outer leaflet of IMM (therefore located in the intermembrane space)
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What important reaction is cytochrome c also involved in?
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Apoptosis
In apoptosis, there are a number of signal transduction pathways focusing on the mitochondria |
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What happens if signals are telling the mitochondria that apoptosis is necessary?
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Transition pores are formed, cytochrome c is released into the cytoplasm, proteolytic enzymes called caspases are released into the cytoplasm and destroy cell.
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What type of reaction is the ATP synthesis reaction?
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It is an ENERGONIC reaction, therefore requiring the INPUT of energy
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What is ATP synthesis mediated by?
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A protein complex in the IMM
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What are some names for this complex in the IMM that mediates ATP synthesis?
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COmplex V
Proton translocating ATP synthase Fo/F1 ATP synthase ATP synthase |
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What does the conservation of energy NOT require?
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covalent intermediates
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What is the chemiosmotic hypothesis?
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Protons are pumped across the IMM during the ETC, and a gradient of energy store is utilized in ATP synthesis when H= come back through ATP synthase
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What are 5 pieces of evidence that support the chemiosmotic hypothesis?
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1-Oxidative phosphorylation requires an intact membrane
2-The IMM needs to be impermeable to ions 3-Electron transport is associated with transport of H+ out of matrix 4-Reconstituion studies 5-Existence of Uncoupling agents |
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How does the requirment of an intact membrane for oxphos validate the chemiosmotic hypothesis?
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If the membrane is leaky, the electrochemical gradient is destroyed, and ATP synthesis cannot occur
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What allows the impermeability of the membrane to ions?
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The membrane is formed with cardiolipin (a special "double" phospholipid)
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Why would you want the IMM to be impermeable to ions?
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The gradient cannot be dissipated
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What is the only way ions can pass through the IMM?
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If pores/special transport proteins form during certain circumstances
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What is the membrane potential of the IMM? Why is it this?
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THe membrane potential is negative. Outside is more postiive due to presence of protons
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What is the only thing that connects ATP synthesis and ETC?
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THE PMF (Proton Motive Force)
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Describe the reconstitution studies
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Vesicle were reconstituted with only a double bilayer membrane, bacteriorhodopsin, and ATP synthesis. When light was shone on bacteriorhodopsin, pumps H+ INSIDE VESICLE. As a result, ATP was synthesized in such a way that it was outside vesicle. Measured ATP levels in solution. Increased after light stimulation.
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What do uncoupling agents do?
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Dissipate the proton gradient.
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What happens to ETC and ATP synthesis when uncoupling agents are added?
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Normal metabolism occurs, electrons are transported, H+ is pumped but a gradient is NOT kept up. Consequently, there is NO ATP synthesis.
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What are two examples of uncouplers?
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DNP (2,4-Dinitrophenol)
FCCP (Carbonylcyanide-p-trifluoromethoxyphenylhydrazone) |
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How do uncouplers like DNP and FCCP work?
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They have a certain level of membrane solubility/diffusibility and have ionizable groups. They pick up H+ and bring into matrix and therefore dissipate the gradient
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How is the energy released?
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As heat
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What is the difference between oxidative phosphorylation and substrate level phosphorylation?
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Oxphos needs energy input, while Substrate level phos conserves a high energy bond (ex: GTP)
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What is an example of mitochondria that can be uncoupled by hormone induction?
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Brown fat mitochondria
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What type of organism has brown fat?
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Hibernating animals
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Why are there so many mitochondria in the brown fat?
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To generate heat- to recover from hibernation
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How does stimulation by norepinephrine for uncoupling work in brown fat?
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cAMP stimulates PKA which stimulates triacylglyerol lipase, which hydrolyzes triacylglycerols to form free fatty acids. The free fatty acids remove purine nucleotide block of the thermogenin channel. H+ gradient is dissipated, therefore energy is released as heat!
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How do rodents and babies compensate by their lack of shiver response?
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THey have brown fat to produce heat to maintain body temperature
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What is the purpose of the second Nerst equation?
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Determine the free energy stored in the proton gradient
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What is the second Nerst equation?
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DeltaG= 2.3 RT [pHout-pHin] + ZF deltaY
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What is Z?
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Ion charge (+1)
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What is deltaY?
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Membrane potential (negative)
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What are the two proposed mechanisms of proton transport into intermembrane space from the mitochondrial matrix?
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1- Redox Loop Mechanism
2-Proton Pump Mechanism |
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What is a redox loop?
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Involves two carriers
1st picks up H+ and associated electrons, gives e to 2nd carrier,allowing release of H+ into intermembrane space |
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What are the requirments of the first carrier?
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Contains more H+ atoms in its reduced state than its oxidized state (ex CoQ)
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What are the requirements of the second carrier?
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The same number of H+ atoms in the reduced and oxidized state
Must be a PURE electron carrier |
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How does the proton pump mechanism work?
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The cavity of the pump is on the matrix side. As electrons pass through, a transient reduction occurs, changing the conformation so that the pump is facign outwards. As e moves away, it is reoxidized, and the pump reverts to its original conformation.
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What are two requirements of the proton pump?
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1-Potential acid group facing inside and outside of IMM
2-AA side chains involved as proton carriers |
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How does pKa of protein and side chains factor in?
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In membrane pKA of aa side chains is high, therefore they are a weak acid and can pick up protons. Conformational change occurs, and the pKa in of the protein becomes lower. Protein becomes a strong acid, and H+ dissociates.
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What are the two principal domains of ATP synthase?
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Membrane spanning Fo domain
Domain projecting into matrix- F1 |
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What are some properties of Fo?
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Integral membrane protein, contains a proton channel
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How is Fo linked to F1?
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Through gamma and b subunits.
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What is the function of the gamma subunit?
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It acts as a rotor
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What is the function of the b subunit?
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It acts as a stator, holds it anchored stationary to the membrane
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How is the F1 domain arranged?
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With alternating alpha and beta subunits, in a donut organization
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What is the purpose of the beta subunit?
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They are the catalytic subunits
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What is the purpose of the alpha subunit?
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They can bind nucleotides but are NOT CATALYTIC, though they are highly homologous to beta subunits
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How can the association of F1 with Fo be broken?
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Treating the IMM with a low concentration of UREA
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What is the function of F1 when the link with Fo is broken?
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It functions as an ATPase, hydrolyses ATP
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How many alpha and beta sites are there?
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3 each
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What are the other subunits of F1?
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Gamma, delta, epsilon
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Which subunit of F1 initiates the conformational change?
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Gamma
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What is the subunit composition of Fo?
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1a
2b 12c |
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What is the function of the 12 c subunits?
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It functions as the rotor- influx of protons though channel at the a/c interface causes rotation
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How is the rotation acheived?
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By induction of a TORQUE
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What are two proteins that block the proton channel in the a subunit of the Fo domain of ATP synthase?
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Oligomycin (reversibly)
Cyclohexylcarbodimide (irreversibly) |
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What is the difference between blocking proton channels or getting rid of gradient by uncoupling?
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For uncoupling agent, e transport will continue, and since you arent making ATP, energy is dissipated as heat
For protein channel blockers, you still have a proton gradient, which can be used for other things. Therefore electron transport would probably slow down to a certain degree because you're pushing protons out across the IMM against an E/C gradient. The more it builds up, the harder it willbe to pump, therefore transport will be slow. We are trying to maintain a steeper gradient that requires more energy. |
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What are the three beta subunits like at any given moment?
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They are each in different conformational states
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What are the three conformational states that the beta subunit could be in?
|
L- binds substrate ADP + PI loosely, no catalytic activity
T- tight binding of ADP+ Pi- catalytically active O-does not bind either, no catalytic activity |
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How does transition occur between the three states?
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L-> T-> O
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In which state is ATP made?
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T
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In which state is ATP released?
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O
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What are conformational rotations initiated by?
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Rotation of gamma (rotor) subunit
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Where is gamma subunit of F1 attached?
|
To c subunits of Fo and to center of apha and beta hexameric donut of F1
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How does gamma subunit cause the beta conformational changes?
|
The gamma rotor has different faces. When it rotates, depending what face the beta subunit is exposed to, will cause it to assume a particular conformation.
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In solution, what is the Keq of the ATP synthesis reaction?
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10^-5
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When the beta subunit is in the T state, what is the Keq?
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1
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Why is the Keq so much higher for the T state beta subunit?
|
The water of hydration has been stripped away from inorganic phosphate + ADP and the two precursors can itneract directly
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Where exactly in ATP synthesis is the energy required?
|
Not in the actually ATP synthesis reaction, but the free energy is used in the rotational catalysis-> causing conformational changes in beta subunits
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How efficient are ATP synthases?
|
Each ATP synthase can make 100 ATP molecules a second!!!
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What are the substrates of ATP synthesis?
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ADP and Pi
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How is Pi transported into the mitochondria?
|
A Pi/H+ symport
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What is an example of a protein BESIDES ATP SYNTHASE that uses the PMF to function?
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Pyruvate translocator
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What is another protein in ATP synthesis that pyruvate translocase is analogous to?
|
The Pi/H+ symport
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How is ADP transported into the mitochondira?
|
ADP/ATP translocator
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How does the ADP/ATP translocator work?
|
The transporter has 1 binding site
If transporter is facing in, there is more ATP than ADP and it competitively binds, causing a conformational change. The transporter is now facing out, but now there is more ADP than ATP and the ADP competes for binding, kicking the ATP out and the transporter can go back in, bringing in ADP to the cell to act as a substrate for ATP synthesis |
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What is the net charge of ATP?
|
-4
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What is the net charge of ADP?
|
-3
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How does the charge of the IMM drive ATP out?
|
SInce ATP is more negatively charged than ADP, and the inside of the IMM is more negative than the intermembrane space, it will preferentially kick out the ATP
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Why are mitochondria important?
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-Major site of ATP synthesis
-Regulators of Apoptosis -Major site of free radical generation |
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Which cell types do NOT use mitochondria as the rmajor site of ATP synthesis?
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Tumor cells
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How does the mitochondria induce apoptosis after integrating all the death signals?
|
Leakage of cyt c to the cytosol, which occurs through the MITOCHONDRIAL PERMEABILITY TRANSITION PORE. Caspases are activated, resulting in cell DEATH.
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Where are free radicals significant?
|
They play an important role in the pathogenicity of a variety of diseases/conditions: neurodegenerative, aging
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What are three examples of free radicals?
|
Superoxide anion,
Hydrogen peroxide Hydroxyl radical |
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Which free radical is the most damaging and where does it cause its damaging effects?
|
The hydroxyl radical damages lipids, proteins, nucleic acids
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How are free radicals formed?
|
During ETC, some electrons are leaked, resulting in the formation of these toxic byproducts
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How is the superoxide radical formed?
|
Electrons are leaked between Complex 1 and 3, from CoQ and The CoQ free radical intermediate (CoQH) can pass e to O2 to form the superoxide radical.
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How is the superoxide radical neutralized?
|
MnSOD (Superoxide Dismutase) forms H2O2 and GPX (glutathione peroxidase) converts this to water
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What happens to the hydrogen peroxide in the presence of Fe2+?
|
Converts to hydroxyl radical (Fenton reaction)
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What is the only way to extend lifespan?
|
Reduce caloric intake by 30-40%
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What is resveratol?
|
A component in wine that mimics caloric deprivation, activating enzymes SIRT3 and SIRT4 that make the mitochondria function more efficiently
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What is the ubiquitin scanning system?
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Detects abnormal proteins and destroys them. But in agiing, this system decreases, and therefore aging may be due to the damage of ROS (reactive oxygen species)
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