18-20: OX. PHOSPHORYLATION Flashcards
Chemiosmosis
-mechanism by which ox. ph occurs
-e transfer and ATP synthesis are coupled by H+ gradient across IMM
-e from reduced cofactors (NAD/FADH) passed through a series of protein carriers in ETC; results in H+ pumpd from matrx to IMS
-[H+] in matrx decreases which sets up H+ gradient across IMM
(IMM is impermeable to H+ so they have to be pumped through proteins as result of e passage)
-this generates electric field (matrix -ve) = proton motive force (PMF)
-flow of H+ back into matrix goes through ATP synthase which drives ATP synthesis
ETC - e transport chain
- e from NADH passed through 3 carriers to oxygen
- complex I, III, IV
- in between there’s 2 mobile carriers (ubiquinone Q and cytochrome c, a small soluble protein)
- complexes are free to diffuse in the membrane
- e will flow in order that they do due to difference in reduction potential
- e passed on to carriers w/more +ve redox potential (higher affintiy) as they flow down an energy gradient
- free energy released is used to generate the proton gradient
Complex I: NADH-Q-OXIDOREDUCTASE
- v.large; has multiple subunits; L-shaped molecule
- accepts 2 e from NADH which becomes oxidised
- 2e passed to Q, will be reduced
- 2 types of e carriers: 1) flavin mononucleotide FMN can accept 2e and 2H+; 2) iron-sulfur clusters accepts 1e, has cysteine residues, up to 10 FeS clusters, e passed one at a time
- e can pass through space but carriers must be <15Å apart
-as result of e passage, 4H+ pumped form matrix to IMS; involves conformational changes to open channels (not quite understood)
ubiquinone (Q)
- first mobile e carrier
- quinone ring (2 keto groups) + long hydrocarbon chain (up to 10 isoprene units); v. hydrophobic; v. soluble in the membrane; moves rapidly through mitochondrial membranes
- e transfer from Q coupled with H+ binding and released
- QH’ is intermediate semiquinone as it accepts 1e; then accepts 2nd e to give QH2 ubiquinol (keto groups reduced to OH)
Complex III: UBIQUINONE - CYT C OXIDOREDUCTASE
- redox loop mechanism for H+ transfer
- accepts 2e from QH2, passes them to cyt c
- has FeS cluster; Fe ligated by 2His and 2Cys
- has 3 haem groups (bH, bL, c1) all in different environments
- has 2 binding sites for QH2 (Qo near IMS; Qi near matrix)
Q cycle
- needed because QH2 carries 2e but cyt c can only accept 1e
- 2xQH2 bind consecutively; each passes 2e + 2H+ (released to IMS)
- in 1x Q cycle, 2QH2 are oxidised to 2Q; 1Q reduced to 1QH2
cytochrome c
- small soluble protein that diffuses in IMS
- has haem group which can accept 1e
- has binding site on complex III where it accepts 1e, becomes reduced and diffuses to BS on complex IV where it donates its e and is oxidised
complex IV: cytochrome c oxidase
- catalyses the reduction of O2 to H2O –requires 4e
- 2 haem groups (A and A3) accept 1 e
- 2 copper centres (CuA and CuB)
- for 2e from NADH/FADH2, 2H+ pumped to IMS
- 4 H+ not contributing to proton gradient; needed to produce water
complex II: succinate dehydrogenase
- membrane bound enzyme of TCA cycle passes 2e from FADH2 to Q to QH2
- no H+ are pumped to IMS
- ETF (e transferring flavoprotein) also passes e to Q from FADH2 produced from fatty acid oxidation
NADH vs FADH2 proton pump
NADH - 10H+ total per 2e
- comp I 4H+
- comp III 4H+
- comp IV 2H+
FADH2- 6H+ total per 2e
-comp III 4H+
-comp IV 2H+
(comp I is bypassed)
uncouplers
- compounds that carry H+ through the IMM to matrix - so they uncouple e transfer from ATP synthesis
- e still flow through ETC to O2 but no ATP is synthesised as proton gradient has been destroyed (energy of H+ gradient is released as heat)
- chemical uncouplers (usually weak acids) e.g. dinitrophenol is toxic
- uncoupling is important physiologically in brown adipose tissue; especially in babies/hibernating animals
ATP Synthase
- large complex protein in IMM; split into 2 parts
- F1 sticks out into matrix; Fo hydrophobic is in IMM
- F1 can be released from Fo by high salt wash
- F1 alone hydrolysis ATP
- Fo needed to transmit energy from proton gradient to F1 and drive reaction to synthesise ATP
- ATP synthesis is powered by PMF (by e transport)
- H+ moving through c ring drive formation of ATP by F1
ATP synthase structure
- 3@ and 3ß subunits arranged alternately in a ring
- @/ß both bind nucleotides but only ß is catalytic
- 3ß subunits have different conformations due to interactions w/ y subunit ( a long @-helix in middle of @/ß ring)
- has 1 x d, y, e
-Fo is a ring of 8-14 identical subunits (c)
- 3 subunits connect Fo to F1; form the ab2 stator
- @ subunit is hydrophobic (next to c-ring) in IMM
- whole stator connected to Fo by @ subunit; to F1 through d
- whole molecule is a rotary motor; rotation drives ATP synthesis
F1
- proton flow around c ring powers rotation and ATP synthesis
- each c has 2@ helices which span the membrane
- in the middle of one @ helix is a -ve residue (glu/asp)
- a subunit has 2 half-channels that don’t span the membrane; half channel on IMS side allows H+ to come in; on matrix side allows H+ to go out
- H+ from H+ rich IMS enters half-channel on c subunit
- Glu/asp (-ve residue) take up H+
- on c subunit near matrix, H+ is released
- for full 360º rotation of c-ring, need 1H+ per c subunit so for mammals 8H+ needed for 360º
Binding change mechanism for ATP synthesis
-proposes there are 3 different conformations of ß subunit binding sites which have different affinities for the nucleotide
loose (L) - binds ADP+Pi
tight (T) - binds ATP v.tightly so will convert ADP+Pi to ATP
open (O) - releases ATP
-they interact differently w/y subunit; ß subunit rotates through 3 different conformations due to rotation of y and c-ring
-S binds to O site
-O becomes L
-ATP is synthesised and site becomes T
-rotation of y causes conformational change releasing ATP
-S binds to a different O site
(in 120º)
