Regulation of PC, Pyruvate Regulation of PC, Pyruvate CarboxylaseCarboxylase • PC is the “anaplerotic” (filling up) reaction for the PC is the “anaplerotic” (filling up) reaction for the

TCA; it is also a G’neo rxn: pyr + ATP + COTCA; it is also a G’neo rxn: pyr + ATP + CO22 oxac oxac + ADP + ADP (Oxidation of 18 of the 20 AA also produces TCA (Oxidation of 18 of the 20 AA also produces TCA ints.)ints.)

• ACoA activates PC; PC requires that ACoA be bound ACoA activates PC; PC requires that ACoA be bound to it in order to be active to it in order to be active

• MR (for G’neo): ACoA is the product of PDH an MR (for G’neo): ACoA is the product of PDH an alternative use of pyralternative use of pyr

• ML: If [ACoA] is high, then pyr is not needed for ML: If [ACoA] is high, then pyr is not needed for PDH, can be “conserved as CHPDH, can be “conserved as CH22O” and converted to O” and converted to GG

• ACoA canACoA cannotnot be converted to G: PDH is irreversible be converted to G: PDH is irreversible AND THERE IS NO BYPASSAND THERE IS NO BYPASS

• MR (for TCA): product of PC is needed to react with MR (for TCA): product of PC is needed to react with ACoA, the fuel of the TCA ACoA, the fuel of the TCA

• ML: if [ACoA] is high, oxac is needed to react with ML: if [ACoA] is high, oxac is needed to react with it, PC must produce oxac. it, PC must produce oxac. (same MRD for G’neo and (same MRD for G’neo and TCA)TCA)

Reduction of the Reduction of the Flavin in Flavin Flavin in Flavin Adenine Adenine Dinucleotide (FAD) Dinucleotide (FAD) and Flavin and Flavin MonoNucleotide MonoNucleotide (FMN)(FMN)

TCA CycleTCA Cycle• Purpose of TCA: Purpose of TCA: oxidizeoxidize C from food molecules C from food molecules• e- from C are transferred to NAD+ or FAD, which e- from C are transferred to NAD+ or FAD, which

carry them to ETcarry them to ET• Compare the oxidation states of C in G, lactic Compare the oxidation states of C in G, lactic

acid, and COacid, and CO22. The average is zero for the C in G . The average is zero for the C in G or lactate, but it is +4 in COor lactate, but it is +4 in CO22..

• Calculating oxidation states for C atoms, based Calculating oxidation states for C atoms, based on atoms or groups on atoms or groups bonded to thembonded to them::

• 1. Count -2 for =01. Count -2 for =0• 2. Count -1 for -0X (-OH; -OR; -SR)2. Count -1 for -0X (-OH; -OR; -SR)• 3. Count +1 for -H3. Count +1 for -H• 4. Count nothing for ANY/ALL C4. Count nothing for ANY/ALL C• 5. sum of above counts, plus that for the C must 5. sum of above counts, plus that for the C must

equal the charge shownequal the charge shown

• Citrate SynthaseCitrate Synthase: oxaloacetate must bind to CS : oxaloacetate must bind to CS and change its conformation in order for ACoA to and change its conformation in order for ACoA to bind. Otherwise, if CS had only one, active bind. Otherwise, if CS had only one, active conformation, ACoA could bind and: ACoA + H2O conformation, ACoA could bind and: ACoA + H2O acetate + CASH. acetate + CASH.

• To “reverse” this, To “reverse” this, 22ATP equivalents are ATP equivalents are consumed, wasted.consumed, wasted.

• Conversion of pyruvate to COConversion of pyruvate to CO22

• The #2 C of pyruvate is released as COThe #2 C of pyruvate is released as CO22 100% in 100% in the “the “2nd2nd turn” of the TCA (0% in 1st) turn” of the TCA (0% in 1st)

• The third C of pyr is released 50% in the “3rd The third C of pyr is released 50% in the “3rd turn” of TCA and the other 50% will be equally turn” of TCA and the other 50% will be equally distributed among the 4 C’s of oxac (12.5% X 4). distributed among the 4 C’s of oxac (12.5% X 4). One half of this total (the 2 end C’s) will be One half of this total (the 2 end C’s) will be released in each round: 25% in the 4th turn; released in each round: 25% in the 4th turn; 12.5% in 5th; etc...12.5% in 5th; etc...

Pyruvate dehydrogenase Pyruvate dehydrogenase (PDH)(PDH)

Pyruvate dehydrogenase Pyruvate dehydrogenase (PDH)(PDH)• In E Coli, a 60 subunit “multi enzyme” complex In E Coli, a 60 subunit “multi enzyme” complex of 4.6 X 10of 4.6 X 1066 g/mol. g/mol.

• Regulation:Regulation:• 1. ACoA can donate its acetyl group to the 1. ACoA can donate its acetyl group to the

“acetyl group carrier” of PDH, preventing it from “acetyl group carrier” of PDH, preventing it from accepting an acetyl group from pyruvateaccepting an acetyl group from pyruvate

• 2. NADH can donate an e-pair to (one of) the “E-2. NADH can donate an e-pair to (one of) the “E-pair carrier(s)” of PDH, preventing it from pair carrier(s)” of PDH, preventing it from accepting an e-pair from pyruvateaccepting an e-pair from pyruvate

• MR: NADH and ACoA are direct products of PDHMR: NADH and ACoA are direct products of PDH• ML: When [NADH] and/or [ACoA] is/are high, ML: When [NADH] and/or [ACoA] is/are high,

there is not a need to produce more. there is not a need to produce more. • Also, pyr isn’t needed as a fuel, can be Also, pyr isn’t needed as a fuel, can be

conserved as CHconserved as CH22O: PDH is irreversible AND O: PDH is irreversible AND has has no bypassno bypass..

Product Inhibition of PDHProduct Inhibition of PDH

PDH Regulation by PDH Regulation by phosphorylation/dephosphorylaphosphorylation/dephosphorylationtion

PDH Regulation by PDH Regulation by phosphorylation/dephosphorylatiophosphorylation/dephosphorylationn• The enzyme (PDH) is The enzyme (PDH) is inactivatedinactivated when Pi is added when Pi is added

to one of its residues in a reaction catalyzed by to one of its residues in a reaction catalyzed by PDH KinasePDH Kinase. This . This kinasekinase is activated by the is activated by the products of PDH, NADH and ACoA (MR, ML above). products of PDH, NADH and ACoA (MR, ML above).

• The The KinaseKinase is inhibited (and so PDH is “activated”) is inhibited (and so PDH is “activated”) by:by:

• 1. pyruvate: MR: direct substrate of PDH. ML: 1. pyruvate: MR: direct substrate of PDH. ML: when [pyr] is high, there is plenty for use as fuel or when [pyr] is high, there is plenty for use as fuel or for for storage as FAsstorage as FAs..

• 2. ADP: MR: PDH feeds fuel to TCA which pushes 2. ADP: MR: PDH feeds fuel to TCA which pushes ET and ATP production in OP. ML: when [AOP] is ET and ATP production in OP. ML: when [AOP] is high, ATP is being consumed and ATP production is high, ATP is being consumed and ATP production is needed.needed.

PDH Regulation by PDH Regulation by phosphorylation/dephosphorylaphosphorylation/dephosphorylationtion• 3. Ca3. Ca2+2+: MR: Ca: MR: Ca2+2+ is released in a muscle cell is released in a muscle cell

in response to a nerve impulse and is required in response to a nerve impulse and is required to trigger protein movement in muscle to trigger protein movement in muscle contractioncontraction. This consumes ATP ML: “same” . This consumes ATP ML: “same” as high [ADP]: need for ATP production.as high [ADP]: need for ATP production.

• CaCa2+2+ also activates PDH phosphatase also activates PDH phosphatase (PDHPase) so that PDH gets dephosphorylated (PDHPase) so that PDH gets dephosphorylated and activated.and activated.

• G’neo consumes TCA ints, as does production G’neo consumes TCA ints, as does production of some AAsof some AAs

The TCA is a The TCA is a Major Major Intersection Intersection of Metabolismof Metabolism

• Numerous Numerous compounds are compounds are produced from produced from or converted to or converted to TCA TCA intermediatesintermediates

Electron Transport,Electron Transport, ET ET• ET: the oxidation of NADH and FADHET: the oxidation of NADH and FADH22 as e as e-- are passed are passed

to Oto O22..• NETNET reaction: reaction: 2NADH + O2NADH + O22 + 2H + 2H++ 2NAD 2NAD++ + 2H + 2H22O. O. • This occurs in three multistep rxns catalysed by large, This occurs in three multistep rxns catalysed by large,

multisubunit, multisubunit, integral membrane multienzyme integral membrane multienzyme complexescomplexes of the inner mitochondrial membrane, of the inner mitochondrial membrane, which surrounds the which surrounds the matrixmatrix, where the TCA occurs. , where the TCA occurs.

Mitochondrion, Matrix, Inner Mitochondrion, Matrix, Inner MembraneMembrane

MitochonriMitochonrialalMembranMembraneses• Protein is a major Protein is a major

structural and structural and functional functional component of component of membranes.membranes.

• Molecules up to 10-Molecules up to 10-kD permeate the kD permeate the outer membraneouter membrane

• The inner The inner membrane is about membrane is about 75% protein, about 75% protein, about 25% phospholipid 25% phospholipid by weight by weight

ET and OP are Tightly ET and OP are Tightly CoupledCoupled• EachEach of the three multienzyme complexes of the three multienzyme complexes

supplies energy for oxidative phosphorylation to supplies energy for oxidative phosphorylation to produce one ATP.produce one ATP.

• Each of the 3 complexes acts as a “proton pump” Each of the 3 complexes acts as a “proton pump” expelling Hexpelling H++ from the matrix. from the matrix.

• The HThe H++ gradientgradient (high [H (high [H++] outside the matrix ] outside the matrix and low [Hand low [H++] inside is) the ] inside is) the common common intermediateintermediate which which couplescouples ET and oxidative ET and oxidative phosphorylation, OP.phosphorylation, OP.

Net Reactions of ET Net Reactions of ET ComplexesComplexes• NADHDH: 2NADH + 2HNADHDH: 2NADH + 2H++ + 2Q + 2Q 2NAD 2NAD++ + 2QH + 2QH22

• Cytochrome Reductase net reaction: 2QHCytochrome Reductase net reaction: 2QH22 + 4 cyto c (Fe+ 4 cyto c (Fe3+3+ ) ) 2Q + 4H 2Q + 4H++ + 4 cyto c (Fe + 4 cyto c (Fe2+2+))

• Cytochrome Oxidase net reaction: 4 Cytochrome Oxidase net reaction: 4 cyto c (Fecyto c (Fe2+2+) + 4H) + 4H++ + O + O22 4 cyto c (Fe 4 cyto c (Fe3+3+ ) + 2H ) + 2H22O.O.

The actual e- transport is to and from: The actual e- transport is to and from: flavin ring, Fe ions in Fe-S centers and flavin ring, Fe ions in Fe-S centers and

hemes of cytochromes, Coenzyme Q, and hemes of cytochromes, Coenzyme Q, and Cu ions.Cu ions.

Iron-Sulfur ClustersIron-Sulfur Clustersof Iron-sulfur Proteinsof Iron-sulfur Proteins

In NADHDH In NADHDH (aka NADH-Q (aka NADH-Q oxidoreductasoxidoreductase) the major e) the major free energy free energy release is in erelease is in e-- transfer to transfer to flavin (FMN). flavin (FMN). So eSo e-- from from FADHFADH22 of TCA of TCA enter “below” enter “below” this point and this point and only 2 ATP only 2 ATP are produced.are produced.

EEoo’, the Standard Reduction ’, the Standard Reduction PotentialPotential

• The The reduction potential reduction potential indicates the tendency to indicates the tendency to accept eaccept e--(s); the more (s); the more the E the Eo’o’, the greater the , the greater the tendency. For example: tendency. For example:

• succinate + COsuccinate + CO22 + 2 e + 2 e-- αα-kg-kg EEo’o’ = -0.67V = -0.67V

• NADNAD++ + H + H++ + 2 e + 2 e-- NADH NADH EEo’o’ = -0.32V = -0.32V

• NADNAD++ has more has more (less negative) E (less negative) E00’’ and a greater and a greater

tendency to accept etendency to accept e-- than does succinate CO than does succinate CO22. .

• If all reactants and products were present at 1M If all reactants and products were present at 1M (except H(except H++: pH = 7), : pH = 7), αα-ketoglutarate would give up 2 e-ketoglutarate would give up 2 e-- to form succinate + CO to form succinate + CO22 and NAD and NAD++ would accept the 2 would accept the 2 ee--. .

• Since the first reaction is reversed, its ESince the first reaction is reversed, its Eo’o’ becomes becomes + 0.67 v, which added to –0.32 v for the 2+ 0.67 v, which added to –0.32 v for the 2ndnd rxn gives rxn gives ΔΔEEo’o’ = +0.35 v. = +0.35 v.

Free Energy and Reduction Free Energy and Reduction PotentialPotential

• ΔΔG°’ = -nF(G°’ = -nF(ΔΔEEoo’) ’) • = -2mole(96.5kJl/v mol)(+0.35 v)= -2mole(96.5kJl/v mol)(+0.35 v)• = -67.3kJ.= -67.3kJ.• This is enough free energy to couple to This is enough free energy to couple to

GTP formation (+30kJ) and have -37 kJ for GTP formation (+30kJ) and have -37 kJ for the reaction.the reaction.

• (F = 96.5kJ/v mol; (F = 96.5kJ/v mol; n = # mol e- cancelled in balanced rxn) n = # mol e- cancelled in balanced rxn)

The Enzyme Complexes of The Enzyme Complexes of ETET• NADH–Q oxidoreductase (NADHDH):NADH–Q oxidoreductase (NADHDH):

• The 2eThe 2e-- from NADH are passed to from NADH are passed to fflavin lavin mmono ono nnucleotide (FMN) a tightly bound prosthetic ucleotide (FMN) a tightly bound prosthetic group of this 25 subunit complex. group of this 25 subunit complex.

• deltaG°’ is a large negative for this step. deltaG°’ is a large negative for this step. • The 2eThe 2e-- are then passed to a series of are then passed to a series of ironiron – –

sulfursulfur clusters, and then to coenzyme Q. clusters, and then to coenzyme Q. • Q has a long hydrophobic chain (CQ has a long hydrophobic chain (C5050) which ) which

keeps it “dissolved” in the inner membrane in keeps it “dissolved” in the inner membrane in which it moves freely. which it moves freely.

• The 2eThe 2e-- of FADH of FADH22 from succinate dehydrogenase from succinate dehydrogenase are passed through FeS proteins to Q, “below” are passed through FeS proteins to Q, “below” the energy releasing step of NADHDH.the energy releasing step of NADHDH.

The Enzyme Complexes of ETThe Enzyme Complexes of ET

• The electrons of QHThe electrons of QH22 are passed are passed oneone at a at a timetime to cytochrome c (cyto c) by the to cytochrome c (cyto c) by the cytochrome reductasecytochrome reductase complex (9 complex (9 subunits), again by way of an Fe-S protein. subunits), again by way of an Fe-S protein.

• Cyto b of this complex contains 2 heme Cyto b of this complex contains 2 heme (Fe ion) groups, heme b(Fe ion) groups, heme bLL and heme b and heme bHH, , which act in passing an e- from one QHwhich act in passing an e- from one QH..-- to to another, producing Q:another, producing Q:2-2-. .

• 4H4H++ ions are also released outside the ions are also released outside the matrix by QHmatrix by QH2 2 in this process, while 2Hin this process, while 2H++ are bound inside the matrix by Q:are bound inside the matrix by Q:2-2-. See . See the Q cycle, below.the Q cycle, below.

Cytochrome ReductaseCytochrome Reductase• Cytochrome Reductase:Cytochrome Reductase:• has 3 subunits that are has 3 subunits that are

the major actors: the major actors: cytochrome b, cytochrome b, cytochrome ccytochrome c11, and an , and an iron-sulfur protein (ISP)iron-sulfur protein (ISP)

• has 2 binding active has 2 binding active sites for Q binding: sites for Q binding:

• Qo, which is toward the Qo, which is toward the oouter side of the lipid uter side of the lipid bilayer (where bilayer (where stigmatellin is bound) stigmatellin is bound) between the Fe-S between the Fe-S cluster and heme bL; cluster and heme bL; and and

• Qi which is toward the Qi which is toward the iinner (matrix) side. nner (matrix) side.

Cytochrome ReductaseCytochrome Reductase• Both Qo and Qi are in the Both Qo and Qi are in the

portion of the protein that portion of the protein that spans the inner spans the inner membrane and is membrane and is surrounded by the long – surrounded by the long – CHCH22 – chains of the lipid – chains of the lipid bilayer in which Q moves.bilayer in which Q moves.

• The reactions catalyzed The reactions catalyzed by cytochrome reductase, by cytochrome reductase, the “the “Q cycleQ cycle” involve ” involve passing epassing e-- from QH from QH22 to to cyto c by way of the ISP cyto c by way of the ISP and cyto c1, and from and cyto c1, and from one one QHone one QH..-- to another to another by way of hemes bby way of hemes bLL and and bbHH, producing Q:, producing Q:2-2-..

The Q Cycle: Cycle 1The Q Cycle: Cycle 1• 1. At Qo, QH1. At Qo, QH22 donates 1e donates 1e--

to the ISP, which donates to the ISP, which donates it to cyto cit to cyto c11, which passes , which passes it to cyto c; QHit to cyto c; QH22 also also donates its 2Hdonates its 2H++ to the to the aqueous medium outside, aqueous medium outside, producing Qproducing Q.-.- ( (noticenotice “dot” and “negative “dot” and “negative charge”)charge”)

• 2. Q2. Q.-.- donates 1e donates 1e-- to heme to heme bbLL which passes it to heme which passes it to heme bbHH; Q is released from Qo; Q is released from Qo

• 3. Q binds at Qi, accepts 3. Q binds at Qi, accepts the 1ethe 1e-- from b from bHH, and , and reverts to Qreverts to Q.-.-..

• Net: QHNet: QH22 + cyto c (Fe + cyto c (Fe3+3+) ) QQ.-.- + cyto c (Fe + cyto c (Fe2+2+) + ) + 2H2H++ (outside) (outside)

Q Cycle: Cycle 2Q Cycle: Cycle 2• 1. 1. AnotherAnother QH QH22 goes goes

through steps 1 and 2 of through steps 1 and 2 of cycle 1, but in step 3 the cycle 1, but in step 3 the electron from belectron from bHH is is donated to Qdonated to Q.-.- at Qi. at Qi. When this Q:When this Q:22-- binds 2H binds 2H++ on the on the matrixmatrix side, one side, one of the 2QHof the 2QH22’s that was ’s that was consumed is consumed is regenerated.regenerated.

• Net for cycle 2: Net for cycle 2:

• QQ.-.- + cyto c (Fe + cyto c (Fe3+3+) + 2H) + 2H++ (matrix) (matrix) Q + cyto c Q + cyto c (Fe(Fe2+2+) + 2H) + 2H++ (outside) (outside)

Q Cycle Net ReactionQ Cycle Net Reaction• Sum for cycle 1 + cycle 2:Sum for cycle 1 + cycle 2:• 1: QH1: QH22 + cyto c (Fe + cyto c (Fe3+3+) ) Q Q.-.-

+ cyto c (Fe+ cyto c (Fe2+2+) + 2H) + 2H++ (outside) (outside)• 2: Q2: Q.-.- + cyto c (Fe + cyto c (Fe3+3+) + 2H) + 2H++ (matrix) (matrix)

Q + cyto c (FeQ + cyto c (Fe2+2+) + 2H) + 2H++ (outside) (outside)• Net:Net:• QHQH22 + 2 cyto c (Fe + 2 cyto c (Fe3+3+) + ) + 2H2H++ (matrix) (matrix)

Q + 2 cyto c (Fe Q + 2 cyto c (Fe2+2+) + ) + 4H4H++ (outside) (outside)

• When QHWhen QH22 binds at Qo, it is in proximity to binds at Qo, it is in proximity to the ISP, donates ethe ISP, donates e-- to it. Then the to it. Then the conformation changes, placing Qconformation changes, placing Q.-.- in in proximity to heme bproximity to heme bHH..

The Enzyme Complexes of ETThe Enzyme Complexes of ET• Four eFour e-- from 4 cyto c (Fe from 4 cyto c (Fe2+2+) are ) are

passed to Opassed to O22 (along with 4H (along with 4H++, , producing 2Hproducing 2H22O) by O) by cytochrome cytochrome oxidaseoxidase (8 subunits). (8 subunits).

• An important point here is that OAn important point here is that O22 binds between Febinds between Fe2+2+ and Cu and Cu++ and and 4 e4 e-- are passed to Oare passed to O22 almost at almost at once. Three of the eonce. Three of the e-- come from come from the changes in the oxidation the changes in the oxidation states of Fe (to 4+) and Cu to states of Fe (to 4+) and Cu to (2+). The fourth comes from a (2+). The fourth comes from a tyrosine side chain hydroxyl tyrosine side chain hydroxyl (forming an oxide radical).(forming an oxide radical).

• The rest involves entry of eThe rest involves entry of e--‘s ‘s and Hand H++’s to form H’s to form H22O and return O and return Fe and Cu to FeFe and Cu to Fe2+2+ and Cu and Cu++..

Cytochrome OxidaseCytochrome Oxidase• OO22 binds between Fe binds between Fe2+2+ and Cu and Cu++ and 4 e and 4 e-- are passed to are passed to

OO22 almost at once. Three of the e almost at once. Three of the e-- come from the come from the

changes in the oxidation states of Fe (to 4+) and Cu to changes in the oxidation states of Fe (to 4+) and Cu to (2+). The fourth comes from a tyr side chain hydroxyl (2+). The fourth comes from a tyr side chain hydroxyl (forming an oxide radical), which also donates its H(forming an oxide radical), which also donates its H++..

Cytochrome OxidaseCytochrome Oxidase• An eAn e-- from cyto c and an H from cyto c and an H++ from the from the

matrix return tyr (Y) to the –OH form.matrix return tyr (Y) to the –OH form.• Another HAnother H++ converts the OH converts the OH-- on the Cu on the Cu2+2+

to water. to water.

Cytochrome OxidaseCytochrome Oxidase• Another eAnother e-- converts Fe from 4+ to 3+ and an H+ converts Fe from 4+ to 3+ and an H+

converts Oconverts O22-- to OH to OH--..

• Then another HThen another H++ produces water and two more e produces water and two more e-- return Fe to 2+ and Cu to 1+. Ready to go again! return Fe to 2+ and Cu to 1+. Ready to go again!

Proton PumpingProton Pumping• It is obvious how the Q cycle can contribute to an It is obvious how the Q cycle can contribute to an

HH++ gradient. Another mechanism for gradient. Another mechanism for ET enzymesET enzymes is is the proton pump:the proton pump:

• 1: nH1: nH++ bind to enzyme side chains on the matrix bind to enzyme side chains on the matrix side of the inner membrane. side of the inner membrane.

• 2. E accepts e2. E accepts e--, providing energy to cause a , providing energy to cause a change in conformational that puts the side chains change in conformational that puts the side chains outside.outside.

• 3. H+ is released 3. H+ is released outsideoutside the matrix. the matrix.• 4. E releases e4. E releases e-- and reverts to original and reverts to original

conformationconformation

Proton PumpingProton Pumping• The 4 steps in pumping HThe 4 steps in pumping H++ above above mustmust occur in occur in

order: can’t have conformational change in step 2 order: can’t have conformational change in step 2 without Hwithout H++ bound bound andand e e-- accepted; can’t revert accepted; can’t revert conformation in step 4 without releasing Hconformation in step 4 without releasing H++ in step in step 3 3 andand e- in step 4. e- in step 4.

• Why is energy release of redox rxns needed at step Why is energy release of redox rxns needed at step 2 rather than for conformational change in step 4? 2 rather than for conformational change in step 4? Step 2 is where HStep 2 is where H++ moves from low [H moves from low [H++] to high ] to high [H[H++]: energy consumed]: energy consumed

• How can HHow can H++ bindbind where [H where [H++] is ] is lowlow and and be be releasedreleased where [H where [H++] is ] is highhigh? The basic group of ? The basic group of the side chain is positioned the side chain is positioned on the insideon the inside where its where its bound Hbound H++ can form an H-bond or ionic bond with can form an H-bond or ionic bond with another side chain. Then, when the conformation another side chain. Then, when the conformation changes, these groups move away from each changes, these groups move away from each other. other.

• It is bonded to 2 groups on the inside and 1 on the It is bonded to 2 groups on the inside and 1 on the outside; part of the energy input is to break this outside; part of the energy input is to break this bond.bond.

Oxidative Phosphorylation, OPOxidative Phosphorylation, OP• OP is the process of ATP synthesis by OP is the process of ATP synthesis by ATP ATP

synthasesynthase, which catalyzes: ADP + Pi , which catalyzes: ADP + Pi ATP. ATP.• It is oxidative in that it is tightly coupled to ET and It is oxidative in that it is tightly coupled to ET and

so to the TCA and other processes that oxidize C so to the TCA and other processes that oxidize C from food.from food.

• ATP synthase has one major portion (FATP synthase has one major portion (Foo) that is ) that is embedded in the inner membrane and another (Fembedded in the inner membrane and another (F11) ) that projects from it into the matrix like a lollipop. that projects from it into the matrix like a lollipop.

ATP SynthaseATP Synthase• When mitochondria are hit with When mitochondria are hit with

ultrasonic waves, they are broken ultrasonic waves, they are broken up into “submitochondrial particles” up into “submitochondrial particles” that are inverted vesicles: the that are inverted vesicles: the lollipops point out. These vesicles lollipops point out. These vesicles can carry out ATP synthesis if can carry out ATP synthesis if supplied with NADH. supplied with NADH.

• When treated with urea, the When treated with urea, the lollipops are dissolved into the lollipops are dissolved into the aqueous medium, the vesicles are aqueous medium, the vesicles are smooth, and they cannot synthesize smooth, and they cannot synthesize ATP. The soluble protein can ATP. The soluble protein can hydrolyse ATP but cannot hydrolyse ATP but cannot synthesize it; this is the “Fsynthesize it; this is the “F11--ATPase”.ATPase”.

• When FWhen F11 is added back to the is added back to the smooth vesicles without urea, the Fsmooth vesicles without urea, the F11 again projects from the membrane again projects from the membrane and the vesicles can synthesize ATP.and the vesicles can synthesize ATP.

ATP SynthaseATP Synthase• The FThe F11 portion has 9 subunits: portion has 9 subunits: αα33ββ33γδεγδε. . • The The αα33ββ3 3 hexamer has hexamer has αα and and ββ subunits alternating like subunits alternating like

the sections of an orange.the sections of an orange.• The gamma (The gamma (γγ) subunit has two long ) subunit has two long αα helical helical

segments projecting like an “axle” up between the segments projecting like an “axle” up between the “wheel” of “wheel” of αα and and ββ subunits. subunits.

ATP ATP SynthaseSynthase

• The FThe Foo part has a subunit composition ab part has a subunit composition ab22ccnn; ; where n= 10 in yeast and ~ 10 in E coli. where n= 10 in yeast and ~ 10 in E coli.

• The c subunits form a “ring” or “barrel” that is The c subunits form a “ring” or “barrel” that is about the same thickness as the inner about the same thickness as the inner membrane and is embedded in it.membrane and is embedded in it.

• The The γγ and, in E coli, and, in E coli, εε subunits are firmly attached to subunits are firmly attached to the c barrel and rotate with it. (the c barrel and rotate with it. (γγ and and δδ in mitochondria) in mitochondria)

ATP ATP SynthasSynthas

e is a e is a MoleculaMolecular Rotary r Rotary MotorMotor

• The a, b2, The a, b2, δδ and and αα33ββ3 3 hexamer subunits are hexamer subunits are stationary, but the c barrel and the stationary, but the c barrel and the γγ subunit are subunit are caused to rotate by the entry of Hcaused to rotate by the entry of H++ into the matrix. into the matrix.

• The gamma subunit is not symmetric. AThe gamma subunit is not symmetric. A different different “face” of it is in contact with each of the “face” of it is in contact with each of the 3 3 αβαβ pairs, forcing the 3 pairs, forcing the 3 ββs to s to alwaysalways be in 3 different be in 3 different conformations.conformations.

• As gamma spins, each As gamma spins, each ββ goes through a sequence of goes through a sequence of conformational changes: O conformational changes: O L L T T O O L L T T

Binding Change Mechanism of ATP Binding Change Mechanism of ATP SynthaseSynthase

• Properties of the 3 conformations of Properties of the 3 conformations of ββ::• O – “open”; low affinity for ATP/ADP: releases ATPO – “open”; low affinity for ATP/ADP: releases ATP• L – “loose”; moderately affinity for binding ADP and L – “loose”; moderately affinity for binding ADP and

PiPi• T – “tight”; tight binding of ATP: can’t release ATPT – “tight”; tight binding of ATP: can’t release ATP• ATP is synthesized on the ATP is synthesized on the ββ subunit that is in the T subunit that is in the T

form. But form. But ATP ATP productionproduction requires that T subunit to be requires that T subunit to be converted to the O form for release of ATP. If T-ATP converted to the O form for release of ATP. If T-ATP reverts to the L form, ADP + Pi will be released.reverts to the L form, ADP + Pi will be released.

• It is the It is the directional sequencedirectional sequence of conformational of conformational changes: changes: O O L L T T O O L L T T etc, that causes etc, that causes ATP production.ATP production.

Evidence for the Binding Change Evidence for the Binding Change MechanismMechanism

• When dissolved FWhen dissolved F11 “particles” ( “particles” (αα3333γδεγδε) were ) were incubated with ADP and Pi in Hincubated with ADP and Pi in H22

1818O, (water in O, (water in which the O is which the O is 1818O) it was found that this O) it was found that this “ATPase” “ATPase” diddid make ATP make ATP, but didn’t release it: , but didn’t release it:

• No ATP product was measured in the solution No ATP product was measured in the solution but it was observed that the but it was observed that the 1818O from the O from the HH22

1818O was incorporated into PiO was incorporated into Pi• HOW? FHOW? F1 1 catalyzes these reactions: catalyzes these reactions: • 1. ADP + Pi 1. ADP + Pi ATP ATP• 2. ATP + H2. ATP + H22

1818O O ADP + ADP + 1818O-PiO-Pi• FF11 was required for this to occur was required for this to occur• ADP was also requiredADP was also required

Rotational Motion from HRotational Motion from H++ FlowFlow

Rotational Rotational Motion from Motion from

HH++ Flow Flow

• An HAn H++ binds to the asp (-CH binds to the asp (-CH33COCO22--) side chain of a c ) side chain of a c

subunit in the cytosolic half-channel between the subunit in the cytosolic half-channel between the a and c subunits. (This is a water-filled channel a and c subunits. (This is a water-filled channel on the intermembrane-space side). on the intermembrane-space side).

• Upon accepting the HUpon accepting the H++, this c subunit changes , this c subunit changes conformation so as to conformation so as to pushpush the c barrel to rotate the c barrel to rotate this subunit “out” from contact with the this subunit “out” from contact with the aa subunit subunit and into contact with the lipid bilayer.and into contact with the lipid bilayer.

• A c subunit in the –CHA c subunit in the –CH33COCO22HH form moves into form moves into contact with the matrix half-channel, where it can contact with the matrix half-channel, where it can release Hrelease H++ into the low [H into the low [H++] environment there.] environment there.

• Repeating the steps on the previous slide Repeating the steps on the previous slide for each of the 10 c subunits:for each of the 10 c subunits:

• i. each of the c subunits delivers a proton i. each of the c subunits delivers a proton (H(H++) from the outside to the matrix (10H) from the outside to the matrix (10H++ in)in)

• ii. rotates the c barrel one complete turnii. rotates the c barrel one complete turn• iii. causes the synthesis of 3 ATP by the iii. causes the synthesis of 3 ATP by the ’s’s

• ““Complete” net reactions for ET and OP Complete” net reactions for ET and OP (per O(per O22, counting 3ATP per NADH):, counting 3ATP per NADH):

• ET: 20HET: 20H++(in) + 2NADH + 2H(in) + 2NADH + 2H++ + O + O22 2NAD2NAD++ + 2H + 2H22O + 20HO + 20H++(out)(out)

• OP: 20HOP: 20H++(out) +6ADP + 6Pi (out) +6ADP + 6Pi 6ATP + 6ATP + 20H20H++(in)(in)

Factors in c Barrel RotationFactors in c Barrel Rotation• In addition to the change In addition to the change

in conformation of the c in conformation of the c subunit, protonation of subunit, protonation of the asp side chain also the asp side chain also breaks asp’s ionic bond breaks asp’s ionic bond with an arg side chain of with an arg side chain of the a subunit.the a subunit.

• This asp-arg bond holds This asp-arg bond holds the c subunit in contact the c subunit in contact with the cytosolic half-with the cytosolic half-channel; but when it channel; but when it breaks and c “pushes breaks and c “pushes off” the barrel can off” the barrel can rotate.rotate.

Experimental Results that Support the Experimental Results that Support the above Mechanism of ATP Synthaseabove Mechanism of ATP Synthase

• ATP synthesis in ATP synthesis in synthetic vescicles by synthetic vescicles by ATP synthase can also ATP synthase can also be “driven” by be “driven” by bacteriorhodopsin, a bacteriorhodopsin, a well known Hwell known H++ pump. pump.

• This protein under goes This protein under goes a conformational change a conformational change when it absorbs a when it absorbs a photon (just as the ET photon (just as the ET enzymes do using enzymes do using energy from redox).energy from redox).

• The ATP synthesis is The ATP synthesis is light dependent in this light dependent in this system.system.

Experimental Results that Support Experimental Results that Support the above Mechanism of ATP the above Mechanism of ATP

SynthaseSynthase• When the αWhen the α3333 hexamer is immobilized on a slide hexamer is immobilized on a slide and a fluorescent actin filament is attached to and a fluorescent actin filament is attached to either the end of either the end of γγ that attaches to the c-barrel or that attaches to the c-barrel or to the distal end of the c-barrel, there is to the distal end of the c-barrel, there is ATP-ATP-dependentdependent rotation that “swings the filament” rotation that “swings the filament” around. around.

• It moves in slow 120It moves in slow 120 steps @ low [ATP], spins steps @ low [ATP], spins faster at high [ATP]. This emphasizes that rotation faster at high [ATP]. This emphasizes that rotation and ATP hydolysis (and ATP hydolysis (or synthesisor synthesis) are paired.) are paired.

Evidence for the Chemiosmotic Evidence for the Chemiosmotic HHypothesisypothesis for Coupling of EP and for Coupling of EP and

OPOP• The chemiosmotic hypothesis: ET and OP are The chemiosmotic hypothesis: ET and OP are

coupled by the Hcoupled by the H++ gradient. ET produces it by gradient. ET produces it by pumping Hpumping H++ out and OP consumes it by using out and OP consumes it by using the entry of Hthe entry of H++ to produce ATP. to produce ATP.

• 1. an 1. an intact membraneintact membrane that does not allow that does not allow diffusion of Hdiffusion of H++ or ions is required for coupling or ions is required for coupling

• 2. adding acid to the medium of a suspension 2. adding acid to the medium of a suspension of mitochondria will increase synthesis of ATPof mitochondria will increase synthesis of ATP

• 3. ET does cause H3. ET does cause H++ to exit the matrix to exit the matrix

• 4. ET and OP are uncoupled by substances that 4. ET and OP are uncoupled by substances that transport H+ or charge across the inner transport H+ or charge across the inner membrane membrane

Uncouplers of ET and OPUncouplers of ET and OP

• Dinitrophenol (DNP) is an uncoupler: in Dinitrophenol (DNP) is an uncoupler: in the protonated (neutral) form, it can the protonated (neutral) form, it can cross the inner membrane, where it can cross the inner membrane, where it can release Hrelease H++: it carries H+ into the matrix : it carries H+ into the matrix and it and it decreasesdecreases ATP production. (It is ATP production. (It is also toxic.)also toxic.)

Uncouplers of ET and OPUncouplers of ET and OP• Valinomycin carries KValinomycin carries K++ across the membrane (into across the membrane (into

matrix) and also decreases ATP synthesis. matrix) and also decreases ATP synthesis. How/why? The KHow/why? The K++ decreases the decreases the charge charge differencedifference that is produced by the proton pumps of that is produced by the proton pumps of ET pumping HET pumping H++ out: K out: K++ carries that + charge back carries that + charge back in.in.

Uncouplers of ET and OPUncouplers of ET and OP

• ET and Op ET and Op are also are also uncoupled uncoupled for heat for heat production in production in brown fat brown fat cells in infant cells in infant mammals in mammals in nonshivering nonshivering thermogenesthermogenesisis

Regulation of ETRegulation of ET ( (andand OP OP andand TCA) TCA)

• The rate of ET is controlled by [ADP]; ET The rate of ET is controlled by [ADP]; ET rate increases when [ADP] inceases. But no rate increases when [ADP] inceases. But no enzyme of ET binds ADP or is regulated by enzyme of ET binds ADP or is regulated by ADP. ADP.

• The [ADP] controls the OP rate by its effect The [ADP] controls the OP rate by its effect on the rate at which ADP binds to on the rate at which ADP binds to subunits subunits of ATP synthase: a [S] effect on this E. of ATP synthase: a [S] effect on this E.

• The rates of ET and the TCA are controlled The rates of ET and the TCA are controlled by the OP rate: all three processes go at by the OP rate: all three processes go at proportional rates. proportional rates.

How OP Controls the Rates of ET and How OP Controls the Rates of ET and TCATCA

• OP consumes the H+ gradient, which is the OP consumes the H+ gradient, which is the product of ET. By doing this, it removes the product of ET. By doing this, it removes the product inhibition effects of the H+ gradient product inhibition effects of the H+ gradient on ET.on ET.

• ET in turn consumes NADH, the product of ET in turn consumes NADH, the product of the TCA, and produces NAD+, the substrate the TCA, and produces NAD+, the substrate of the TCA. This removes the effects of of the TCA. This removes the effects of NADH in product inhibition, and increases NADH in product inhibition, and increases [S], increasing the rates of TCA enzymes. [S], increasing the rates of TCA enzymes.

How OP Controls the Rates of ET and How OP Controls the Rates of ET and TCATCA• When the rate of ATP synthase is low, the HWhen the rate of ATP synthase is low, the H++

gradient is consumed slowly and the gradient gradient is consumed slowly and the gradient approaches a maximum.approaches a maximum.

• When the gradient is at max (at rest, when ATP When the gradient is at max (at rest, when ATP consumption is low and [ADP] is low) then net consumption is low and [ADP] is low) then net HH++ release by ET on outside is slowed by the release by ET on outside is slowed by the high [Hhigh [H++] and H] and H++ binding on the inside is slowed binding on the inside is slowed by the low [H+]: ET enzymes by the low [H+]: ET enzymes mustmust “pump” H “pump” H++ in order to catalyze e- transfer.in order to catalyze e- transfer.

• The gradient is the product of ET; when it is The gradient is the product of ET; when it is high ET is slowed (and when it is low, ET goes).high ET is slowed (and when it is low, ET goes).

• When ET is slow, the rate of NADH consumption When ET is slow, the rate of NADH consumption slows, and [NADH] increases. This causes slows, and [NADH] increases. This causes product inhibition effects that slow TCA. And ET product inhibition effects that slow TCA. And ET produces NADproduces NAD++ slowly, [NAD slowly, [NAD++] decreases, and ] decreases, and enzymes of TCA have a lower [S] supply, enzymes of TCA have a lower [S] supply, slowing them.slowing them.

How OP Controls the Rates of ET and How OP Controls the Rates of ET and TCATCA

• When OP is rapid in response to high [ADP], the When OP is rapid in response to high [ADP], the rate of Hrate of H++ entry into matrix increases and [H entry into matrix increases and [H++] ] outside is decreased while [Houtside is decreased while [H++] inside is ] inside is increased: gradient is less. increased: gradient is less.

• When the gradient decreases, the rate of HWhen the gradient decreases, the rate of H++ binding on matrix side is faster (higher Hbinding on matrix side is faster (higher H++ inside) inside) and net release outside is faster (lower Hand net release outside is faster (lower H++ outside) so ET redox reactions are faster: rate of outside) so ET redox reactions are faster: rate of ET is controlled by the level of the HET is controlled by the level of the H++ gradient. gradient.

• When ET is fast, the rate of NADH consumption When ET is fast, the rate of NADH consumption increases, and [NADH] decreases. This decreases increases, and [NADH] decreases. This decreases the product inhibition effects, so TCA can go. the product inhibition effects, so TCA can go. And ET produces NADAnd ET produces NAD++ rapidly, [NAD rapidly, [NAD++] increases, ] increases, and enzymes of TCA have a greater [S] supply, and enzymes of TCA have a greater [S] supply, increasing their rates.increasing their rates.

How OP Controls the Rates of ET and How OP Controls the Rates of ET and TCATCA

• LeChatelier’s Principle effects caused by LeChatelier’s Principle effects caused by [ADP]:[ADP]:

• TCA: ACoA + GDP + Pi + 3NADTCA: ACoA + GDP + Pi + 3NAD++ +FAD +FAD 2CO2CO22 + GTP + 3NADH + FADH + GTP + 3NADH + FADH22

• ET: 20HET: 20H++(in) + 2NADH + 2H(in) + 2NADH + 2H++ + O + O22 2NAD2NAD++ + 2H + 2H22O + 20HO + 20H++(out)(out)

• OP: 20HOP: 20H++(out) +(out) +6ADP6ADP + 6Pi + 6Pi 6ATP + 6ATP + 20H20H++(in)(in)

Electron Pair Electron Pair ShuttlesShuttles

• The NADH produced in The NADH produced in the cytosol (cytoplasm) the cytosol (cytoplasm) in G’lys cannot be in G’lys cannot be transported into the transported into the matrix for ET. But its e- matrix for ET. But its e- pair is transported by the pair is transported by the glycerophosphate shuttle glycerophosphate shuttle in skeletal muscle and in skeletal muscle and brain:brain:

• The NADH is used to The NADH is used to reduce DHAPreduce DHAP

• The glycerol-3-P The glycerol-3-P produced in this reaction produced in this reaction is oxidized back to DHAP is oxidized back to DHAP by an enzyme on the by an enzyme on the outer surface of the inner outer surface of the inner membrane.membrane.

• The FADHThe FADH22 produced in produced in this reaction is this reaction is consumed by ET.consumed by ET.

The Malate-The Malate-Aspartate Shuttle Aspartate Shuttle Produces NADH in Produces NADH in

the Matrixthe Matrix

• The NADH in the The NADH in the cytosol reduces cytosol reduces oxac to malate. oxac to malate. The malate enters The malate enters the matrix and is the matrix and is converted to oxac, converted to oxac, producing NADH.producing NADH.

• The malate is The malate is converted to asp converted to asp for exit to cytosol for exit to cytosol because oxac because oxac doesn’t exit the doesn’t exit the matrix.matrix.

• The amino group The amino group of asp is of asp is transferred to transferred to αα kg kg producing glu, which producing glu, which is transported back is transported back to the matrix to bring to the matrix to bring the amino back.the amino back.

ATP Produced G’lys, PDH, TCA, ET, and OPATP Produced G’lys, PDH, TCA, ET, and OP• G’lys, PDH, and TCA (See 62405):G’lys, PDH, and TCA (See 62405):• Converted G to 6 COConverted G to 6 CO22

• Produced 2 ATP + 2 GTPProduced 2 ATP + 2 GTP• Produced 2 FADHProduced 2 FADH22 + 10 NADH + 10 NADH• When ET consumes the 2 FADHWhen ET consumes the 2 FADH22, OP , OP

produces 4 ATPproduces 4 ATP• When ET consumes the 10 NADH, OP When ET consumes the 10 NADH, OP

produces 30 ATP, for a net of 38 ATP / Gproduces 30 ATP, for a net of 38 ATP / G• When the glyerol-3-P shuttle is used:When the glyerol-3-P shuttle is used:• 4 FADH2 go into ET, and 8 ATP from OP, and4 FADH2 go into ET, and 8 ATP from OP, and• 8 NADH go into ET, and 24 ATP from OP. 8 NADH go into ET, and 24 ATP from OP.

This nets 36 ATP / G This nets 36 ATP / G