BCEM 393 - Oxidative Phosphorylation

oxidative phosphorylation

ETC and proton gradient

electrons from NADH and FADH2 flow down the electron transport chain through a series of exergonic redox reactions

matrix

where the enzymes of the TCA cycle are (except for succinate dehydrogenase)

inner mitochondrial membrane (IMM)

where succinate dehydrogenase and the electron transport chain (ETC) complexes are located

electron transport chain

4 protein complexes (CI-CIV) - all integral membrane proteins

2 mobile e- carriers - ubiquinone (Q) and cytochrome C (cyt C)

flow of e- from NADH and FADH2

overall has a very large free energy release (DG = -220 kJ) over small steps - energy used to pump protons

redox potential (E’^o)

a measure of the electron affinity of a substance

oxidized form

X- loses e-, e- flow from sample cell to the standard cell

negative reduction potential (likes to transfer e-)

oxidized substance has lower affinity for electrons than H2

reduced form

X gains e-, e- flow from standard cell to sample cell

positive reduction potential (likes to receive e-)

oxidized substance has higher affinity for electrons than H2

NADH and FADH2

electron carriers

NADH transfers e- to CI

FADH2 passes e- to CII (less redox potential than NADH) - bypasses CI

iron

all 4 complexes contain

reduction potential of iron depends on the environment

appears in the ETC as Fe-S clusters and heme groups (Heme A)

reduced (Fe3+ + e- —> Fe2+)

copper

in cytochrome c oxidase, in addition to iron

reduced (Cu2+ + e- —> Cu+)

coenzyme Q

ubiquitous quinone, isoprene groups (10 repeating units form a hydrophobic tail), carries protons and electrons

proton pumps

CI, CIII, and CIV

move H+ from matrix to the IMS

FADH2 results in fewer protons pumped than NADH

NADH-Q oxidoreductase (CI)

electrons flow from NADH to flavin mononucleotide to Fe-S clusters to Q to form Q2-

membrane arm - transfer of protons to make QH2

structural change occurs when QH2 dissociates, results in proton ejected

3 additional protons ejected through electrostatic pressure

TOTAL = 4 H+ into IMS

succinate-Q reductase (CII)

succinate dehydrogenase is part of this complex

electron carriers - FAD, iron-sulfur proteins, coenzyme Q

FADH2 remains part of the complex

succinate oxidized to fumarate, ubiquinone reduced to ubiquinol

cytochrome c reductase, Q-cytochrome c oxidoreductase (CIII)

electrons from QH2 pass to cytochrome c, cyt c carries only one e-

CIII structure

subunits include: cytochrome c (heme), cytochrome b (heme bL, heme bH), Rieske iron-sulfur proteins

Q cycle

mechanism for transfer of 2 e- from QH2 to one electron acceptor, cytochrome c

couples electron transfer with pumping of H+ into IMS

first half of Q cycle

• QH2 binds complex III

• two protons from QH2 are released to IMS

• one e- is transferred to cytochrome c (cyt c is reduced and can move to CIV)

• one e- is transferred through cyt b to Q to form Q-* (radical)

• oxidized Q dissociates from CIII

second half of Q cycle

• second QH2 binds CIII

• two protons from QH2 are released to IMS

• one e- is transferred to cyt c (2nd cyt c) - cyt c is reduced and can move to CIV

• one e- is transferred through cytochrome b to Q-* and 2 H+ from the matrix are transferred to make QH2

cytochrome c oxidase (CIV)

1. 2 cyt c reduce Heme a3 and CuB

2. reduced CuB and Heme a3 bind O2

3. O2, Fe, Cu form a peroxide bridge

4. 2 e- from 2 cyt c and 2 H+ from matrix cleave peroxide bridge

5. addition of 2H+ leads to release of 2 H2O

(total of 4 cyt c arriving at complex)

TOTAL = 4 H+ pumped from matrix to IMS

respirasome

organizing enzymes into complexes improves efficiency

close proximity controls e- movement

proton motive force

chemical and charge gradient

pH gradient is 1.4 pH units

voltage gradient is 0.14 V

the IMS is more acidic (more H+)

ATP synthase

a dimer (2 of the same structures)

ATP synthase structure

c ring, a, b2, s, a3, B3, Y, and E

exterior column

a, b2, s (ATP synthase)

hexomeric ring

a3 and B3 (ATP synthase)

central stalk

Y and E (ATP synthase)

B conformations

open, loose, tight

open conformation

B can bind and release substrate

loose conformation

substrate can be trapped in B

tight conformation

B synthesizes ATP

binding change mechanism

Y rotates, causing the conformation of B to change

120^o rotation of Y counter clockwise

subunit a

has two hydrophilic half channels (intermembrane and matrix half-channel), each half channel directly interacts with one c subunit

protons enter through intermembrane half channel and exit through the matrix half channel

glutamate on subunit c

protonated at the intermembrane half channel

protonated form rotates into the hydrophobic environment of the membrane

releases proton in matrix half channel

rotation of c ring rotates Y subunit

c ring rotation

driven by proton motive force

ring cannot rotate in either direction due to Arg residue in subunit a between half channels

rotates clockwise

glycerol-3-phosphate shuttle

shuttle in muscle, moves e- from cytoplasmic NADH into the mitochondrial electron transport chain

IMM is impermeable to NADH/NAD+

yield is only 1.5 ATP because shuttle uses FAD

NADH is oxidized to NAD+ and H+ are accepted by glycerol 3-phosphate which give e- to FADH2

malate-aspartate shuttle

shuttle in heart and liver, moves e- from cytoplasmic NADH into the mitochondrial electron transport chain

IMM is impermeable to NADH/NAD+

NADH oxidized to NAD+ and carried by malate

malate moved against gradient, a-ketoglutarate moved along gradient (antiporter)

rebalances C atoms across the membrane