BIOL 2056 - Mitochondria and Bioenergetics

how do cells get their energy

• sugar

• amino acids

• fatty acids

• sunlight

• methane

• plants generate their energy in chloroplasts

• prokaryotes use bacterial membrane

the mitochondria

• became encapsuled from another organism

• have their own transcription and translation

• have a 16Kd genome which encodes rRNA, tRNA etc

• requires a large SA for the electrical gradient

mitochondrial matrix

• supports the TCA cycle, beta oxidation of fatty acids which is the primary output of NADH

• supports the urea cycle, amino acid synthesis and mitochondrial protein synthesis

• for these reactions we must get substrates IN

• the TCA cycle provides starting materials for:

  • Amino acids

  • Porphrins (heams, chloroplasts)

  • purines and pyrimidines

the mitochondiral membrane

has features which reflects the processes that it supports:

• inner membrane is protein rich with a slightly less protein dense outer memb

• mitochondrial membrane contains very little cholesterol compared to eukaryotes

• high conc of phosphatidylcholine and phosphatidylethanolamine

• low conc of Pi and phosphatidylserine

• has cardiolipin

  • 4 chains, 2 phosphate groups

  • anionic lipid

  • occupies a large volume —> has a small head group and large chains which causes the membrane to curve

outer mitochondrial membrane

• must communicate with the mitochondria

• has an important role in determining how the mitochondria functions

• has porins which make it permeable

mitochondrial porins

• beta barrel like structure

• relatively unselective

• voltage dependent anion channel for the transport of ADP/ATP

• the voltage gating comes from the helical chain

• lined with positive residues for selectivity

• allows ADP/ATP to flow easily

• has two conformations: one where the alpha helix is in the middle (closed) and one where the alpha helix moves out the way (open)

interface to cell

• it has mechanical links to other organelles and the cytoplasm

• regulation of apoptosis —> release of electrochemical gradient regulates cell apoptosis

location

• mitochondria are distributed throughout the cell

• close to the microtubules (colocalised)

• the outer mt membrane contains miro which binds to milton —> milton is attached to dynein/kinesin

• links to myosin will anchor the mt at a certain place

• links to synaptophysin will link the mt to the microtubule

• mt can have reversible disengagement from the microtubule motor in a calcium dependent manor

• miro can also permanently be broken down leaving a mt at a certain location

mitochondrial contact sites

• mt bound to rough ER

• recruit a whole series of molecular components to form contact sites

• contact sites facilitate the exchange of lipids

• efficient coupling of mt and Er by VDAC adaptors

• this supports the metabolic efficiency

energy generation in the inner mitochondrial membrane

• transfers high energy electrons from donor to teh high energy terminal receptor

• and couples this process with the transfer of protons across the bilayer

F1/F0 ATPase

• primary protein for ATP production

• F1 head is an enzyme that converts ADP—>ATP

• driven by motor complex which is driven by electrochemical gradient

ATP/ADP shuttle

• gets substrate into the inner membrane

• open face on one side for ADP

• conformational change coupled with the binding of ATP

• well defined RRRMMM nucleotide binding domain 2×6 transmembrane domains which forms a dimer

shaping membranes

• membrane folds round on itself caused by F1 ATPases

• fluid mosaic model but although the lipids are fluid, it still has a structure which is controlled by lipids and proteins

MICOS

• is a complex that enables tight turning of christae

• drives the membrane invagination

• composed of MIC10 and MIC60

MIC10

• main role is cell membrane sculpting

• 2 transmembrane domains with transmembrane glycine motif and positively charged loop

• forms a large oligomeric complex

• requires cardiolipin

• the glycine disrupts secondary structures and allows efficient packing of the transmembrane domain

• the positive loop recruits cardiolipin to the loop

MIC 60

• forms a contact between the inner mitochondria and the outer mitochondria

• interactions with VDAC, TOM and TIM complex

• important to localising activity in the inner and outer membrane

why so elaborate

allows this process to work more efficiently:

• advantageous for the electrochemical gradient

• ETC is in flatter regions of the cisternae

• creates a very small volume which can generate a large change in proton concentration in a very small region

• localises the F1 ATPases in close proximity

the electron transport chain

• need to be able to capture the energy released

• therefore reactions are broken down into smaller steps

• there’s no fast, explosive release of energy —> at each step the electrons release energy

• the electrons become more positive as they move down the chain

chemiosmotic theory

• high energy electrons are used to generate an electron gradient

• these high energy electrons then utilise the electrochemical gradient to:

  • power molecular motors that drive ATP biosynthesis

  • drive transport of molecules against their concentration gradient

• this is conserved in all organisms

the electrochemical gradient

• is a gradient of protons —> high concentration outside, low concentration inside

• there’s an electrical potential too from the charge

• it doesn’t have to be H+

• units used are kJ/mol

• chemical gradient is deltapH

• electrical gradient is deltaψ and represents the change in charge across a membrane

• it is often derived in protonmotive force (deltap) which has the units of mV

PREDICTING MOEVEMNT OF ELECTRONS

• we can use this equation to derive protonmotive force and predict the moevemnt of electrons where we replace deltap with deltaG

• 

F = the faraday constant

z = charge

Em (denoted the potential at which the pompound is half oxidised and half reduced)

• is the tendency of chemical species to acquire electrons

• large Em is a high affinity, a low Em is a low affinity

• electrons will move towards a larger Em

using the Nernst Equation

• using the above equation we can rewrite our equation for free energy for a given mass action ratio

• which in terms of electrochemical potential is:

• 

NAD+/NADH REDOC CENTRE

FLAVINS

UBIQUINONE

• formed in the membrane and is anchored by a large isoprenoid chain

• ubiquinone —> ubiquinol

FeS CENTRES

CYTOCHROMES

complex 1 - NADH/Ubiquinone oxidoreductase

• NADH is close to the flavin molecule and the electron jumps to the flavin

• it has a transmembrane component and a large extracellular component

• A series of FeS centres that work their way down this long arm and the electron uses these to jump down

  

• however, they are large distance apart so we would expect the reaction to occur slowly due to large activation energy, however, this doesn’t occur due to electrons quantum tunnelling

moving electrons to the UQ binding site

LOCATION OF THE UQ BINDING SITE

• its a 6 membered ring with an isoprenoid chain

• its embedded in the membrane

• UQ binding site relatively hydrophilic in nature

• the transfer of protons from the Tyr/His as Uq essentially pulled out the bilayer

proton pumping of complex 1

• NADH ubiquinone oxidoreductase

• there are 5 diffrenet complexes with an alpha helix running paralell through the membrane

• the transverse helix reaches out across the pumps to the Uq binding site

• the transverse helix couples the conformational change to the energy transfer

• it moves back and forth and couples the conformational change to the energy transfer

  • 4 different transport proteins allow the movement of 4 protons across the membrane

complex 3

• is a ubiquinone/cytochrome c oxidoreductase

• it oxidises ubiquinone and moves the electrons to cytochrome c

• ubiquinone carries 2 electrons and cytochrome c carries 1 electron therefore 1 electron must be stored

• complex 3 has heam groups

Q cycle

• ubiquinone binds to a binding site

• reduction of ubiquinone releases 2 protons onto the p face of the membrane

• 1 electron will move to the FeS centre where it is loaded onto Cyt C

• we now have a free radical and so we need to find a way of storing the electron on the ubiquinone

SOLUTION:

• the electron is shuttled to cytochrome bL to cytochrome bH

• this electron is used to reduce the ubiquinol to ubiquinone

• however, we only have one electron added to UQ so this needs to happen twice so UQ—> UQ- —> UQH2

why do electrons take different paths?

• repositioning of Rieske proteins directs electrons to different redox centres

cytochrome C

• a small molecule with one cytochrome in the middle

• one face has a cavity which is a binding site for electrons

• has a very positive charge

• cardiolipin has a very negative charge so there’s an electrostatic attraction between cyt C and the lipid bilayer

complex 4: cytochrome C reductase

• cytochrome C oxidase enzyme

• reduce cytochrome C to pass electrons down to molecular )2

• adds 4e- to split O2 into water

• 

• the binding site is at the top which passes electrons down to copper centres to haem centres

• couples the loss of energy to the movement of protons

• 4 protons are pumped out, 4 are reacted with molecular oxygen to make water

• has a special haem A3 which also has a copper centre

reduction of O2

• occurs at a binuclear centre (cytA3/CuB)

• O2 binds to cytA3 which splits O22 and forms a double bond

• an additional oxygen is bound to copper B

• requires 4e-

• 2 come from cyt A3 which adopts a 4+ oxidation state, one comes from a tyrosine molecule that becomes a neutral free radical

• cytochrome c then delivers an e- and a H+ which restores the tyrosine molecule

• delivery of another E- reduces cyt to 3+ state and the H is donated to the O atom which forms a hydroxyl group

• further delivery of 2e- and 2H+ restores Cyt A and CuB to their original oxidation state and allows 2 molecules of water to form and dissociate from the complex

• 

pumping protons

• 2 channels

  • D channel: proton pumping pathway

  • K channel: protons for H2O

• there’s a network of charge residues where you can see these protons hopping from one side to another

• 

summary: ETC

ATP generation: F1/F0 ATPase

At this point all the protons have been pumped across the membrane ready for ATP synthesis

• F1 is the site of ATP synthesis

• F0 is the motor unit

• couples delta p driven protein rotation with ATP synthesis

• coupling between F1 and F0 is done by the central stalk

STRUCTURE

• catalytic domain has an alpha and beta subunit

• theres a stalk domain with a gamma delta and epsilon subunit

• A C10 ring with an A subunit which is part of the motor complex

• the peripheral stalk remains constant

• catalytic domains oscillate which is coupled to ATP synthesis

F0 MOTOR

• 2 types of subunit:

  • A subunit: a stator which provides half channels for translocation of proteins across the bilayer

  • C subunit: a rotor, 8 copies in the mitochondria

• couples the movement of protons across the bilayer to the rotation of the central stalk

• the C subunits form a ring with a conserved glutamate in the middle

• there are two half channels and the protos get transferred to the glutamate between them

• the C subunits form a ring of charge and as the rotor rotates the protons get bought round to the second half channel

• they get removed from the Glu by arginine

• the number of C subunits varies across organisms

  • the lower the electrochemical potential requires more protons to move across the bilayer

  • as a result it puts more C subunits into the bilayer which moves more low energy protons across to generate ATP

transmission to F1 via electrical stalk

• processive - as it rotates it pushes against the things that are around it which provides energy for ATP generation

• 3 roughly symetrical domains

• 3 a/b domains

  • 1 ATP

  • 1 ADP + Pi

  • 1 is empty

• the gamma subunit distorts the binding site which pushes the ADP + Pi

• as the stalk rotates it causes a conformational change in the subunits which causes it to be converted from ADP —> ATP

• whilst the stalk rotates the bound ATP undergoes a conformational shape change which leads to the release of ATP

• the same conformational shape change causes the binding site to collapse down round the ADP and Pi

• this allows ADP and Pi to bind to the binding site so it gets converted to ATP

• each rotation generates 3ATP