BIOS 202: Oxidative Phosphorylation

chemiosmotic theory

ATP synthesis is driven by proton concentration gradient

mitochondria structure

two membranes, outer membrane readily permeable to small molecules, inner membrane impermeable to most molecules, including protons, mitochondrial matrix inside inner membrane includes pyruvate dehydrogenase complex, TCA cycle enzymes, fatty acid and amino acid oxidation pathways

NAD-linked dehydrogenases

nicotinamide nucleotide-linked dehydrogenases, remove two hydrogens from substrate, a hydride and a proton, making NADH and H+, NADH is carried from metabolic reaction sites to entrance of respiratory chain

reduction potential meaning

higher reduction potential means that the species will be reduced and the other oxidize

coenzyme Q

Ubiquinone, electron carrier in the respiratory chain, accepts 1 or 2 electrons, can diffuse through the inner membrane, carries both electrons and protons passes electrons from complexes I and II to III

cytochromes

proteins containing a heme group, three types in mitochondria: a, b, c, heme cofactor in c is covalently bound with Cys residue, cytochrome c passes electrons from complex III to IV

iron sulfur proteins

iron associated with inorganic sulfur or Cys instead of a heme group, involved in 1 electron transfers

complex I

NADH dehydrogenase/NADH:ubiquinone oxidoreductase, L shaped, catalyzes the transfer of a hydride from NADH and a proton from the matric to Q, NADH + H+ + Q -> NAD+ + QH2, coupled with transfer of 4 protons from matrix to intermembrane space (against gradient), electron transfer to Q inhibited by amytal, rotenone, and piericidin A

complex II

succinate dehydrogenase, membrane bound, electrons move from succinate binding site to FAD to Fe-S centers, to Q binding site

complex III

cytochrome bc1 complex/ubiquinone:cytochrome c oxidoreductase, transfers electrons from 2 electron carrier Q to single electron carriers (cytochromes) through the Q cycle, coupled with transfer of 4 electrons into intermembrane space from matrix

complex IV

cytochrome oxidase, transferes electrons from cytochrome c to Cu A center in subunit II, then heme a in subunit I, then heme a3-CuB center, then O2, converts O2 to 2H2O

proton motive force

energy stored in proton gradient, two components: chemical potential energy and electrical potential energy, around 200 out of 220 of energy released by oxidizing a mole of NADH is conserved in proton gradient

redox equation of oxidative phosphorylation

NADH + 11H+ (N) + 1/2O2 -> NAD+ + 10H+ (P) + H2O, where N means negative side of proton gradient in the matrix and P means positive side of gradient in the intermembrane space

conditions necessary for ATP synthesis

oxidizable substrate, O2 consumption, ADP and Pi are present

oligomycin and venturicidin

bind to ATP synthase and inhibit them, which also causes inhibition of the respiratory chain

coupling of ATP synthase and electron transfer

if ATP Is stopped, protons are no longer transferred into the matrix by ATP synthase, so respiratory chain will eventually be unfavorable due to high concentration of H+ in intermembrane space

uncouplers

physically disrupting structure can uncouple, 2,4-dinitrophenol (DNP), FCCP, weak acids with hydrophobic properties that can diffuse across membrane and release protons into matrix, ionophores—valinomycin, dissipate electrochemical gradient by allowing inorganic ions across membrane

two compartments of ATP

Fo compartment is embedded in the membrane, has a pore which protons can leak through easily, it is sensitive to oligomycin, F1 is the compartment which catalyzes ATP synthesis, just Fo in a mitochondria will destroy the proton gradient, isolated F1 can catalyze ATP synthesis, F1 also plugs Fo’s proton pore

conformation changes of ATP Synthase

three beta subunits in ATP synthase, all have a catalytic site for synthesis, gamma subunit is associated with one beta subunit so its conformation is different, one subunit binds ATP, one ADP, and one is empty, the subunit with ADP and Pi catalyzes the synthesis of ATP and changes to beta-ATP conformation, which binds ATP very tightly, it then changes to beta-empty conformation, releasing ATP, it will final switch back to beta-ADP and bind new substrate, conformational changes are driven by passage of protons through Fo, which rotates the subunits so a different one comes in contact with gamma, all three conformations must be different at any given time

P/O ratio

hard to measure because exact ATP synthase mechanism is still unknown and O2 is consumed in mitochondria for other things besides oxidative phosphorylation, generally accepted numbers are 10 protons per NADH and 6 for succinate, divide all by number of protons required to run ATP synthase (4ish), get 2.5 and 1.5

translocases and ATP synthase

ATP synthase is intertwined with two translocases, adenine nucleotide translocase—binds ADP3- in intermembrane space and exchanges it with ATP4- in the matrix, favored by proton/electrochemical gradient because it moves more negative change out,—and phosphate translocase—imports H2PO4- and H+ into the matrix

malate aspartate shuttle

in the cytosol, NADH reduces oxaloacetate to malate through malate dehydrogenase, then malate passes into the matrix through the malate-alpha-ketoglutarate transporter, then NAD+ is reduced to NADH and malate is reduced to oxaloacetate by malate dehydrogenase, NADH goes to respiratory chain, then aspartate aminotransferase changes oxaloacetate to aspartate and glutamate to alpha-ketoglutarate, aspartate exits the inner membrane, around 2.5 ATP per NADH

section 19.3 for regulation of oxidative phosphorylation not done

rotenone

inhibits complex 1 by blocking Fe-S sites, prevents electron transfer from complex I to CoQ

antimycin a

inhibits complex 3 by binding, disrupts Q cycle between complexes 3 and 4

oligomycin

blocks protein channel in Fo