Characteristics of metabolic pathways

Aside from its role in energy :

Glycolysis is a good example of a .

Two common characteristics of a metabolic pathway, in general:

1) Each step = a small chemically reasonable change

2) The overall ∆Go is substantial and negative.

1 Energy yield But all this spewing of lactate turns out to be wasteful.

Using as an oxidizing agent could be completely oxidized, to: … CO2 That is, burned.

How much energy released then?

Glucose + 6 O2  6 CO2 + 6 H2O ∆Go = -686 kcal/mole ! Compared to -45 for glucose  2 lactates (both w/o ATP production considered)

Complete oxidation of glucose, Much more ATP But nature’s solution is a bit complicated. The fate of pyruvate is now different 2 From : acetyl-CoA 3 Scores: per glucose 2 NADH 2 ATP pyruvic acid 2 NADH

2 CO2

Krebs cycle Tricarboxylic acid cycle TCA cycle

α keto

succinic Handout 9A acid 3 Acetyl-OCoA O||

CH3 –C –OH + co- A  acetyl~CoA (acetate)

coA acetate group

(vitamin B5) 4 Per glucose FromB glycolysis: acetyl-coA pyruvate Input 2 oxaloacetates 2 NADH 2 ATP pyruvic acid 2 NADH 2 NADH 2 CO 2 NADH 2 2 CO2 citric acid 2 CO2 oxaloacetic acid 6 CO2 Krebs Cycle isocitric acid malic acid

fumaric acid α keto glutaric acid

5 GTP is energetically equivalent to ATP

GTP + ADP  GDP + ATP

ΔGo = ~0

G= guanine (instead of adenine in ATP)

6 acetyl-CoA B Per 7glucose 2 oxaloacetate 2 NADH 2 ATP pyruvic acid 2 NADH 2 NADH 2 NADH 2 FADH2 citric acid 2 NADH oxaloacetic acid 2 CO2 2 CO2 Krebs cycle 2 CO2 isocitric acid malic acid 6 CO2

fumaric acid α keto glutaric acid

succinic acid 7 FAD = flavin adenine dinucleotide

Business end (flavin) ~ Vitamin B2 ribose

adenine -

ribose FAD + 2H.  FADH 2 8 D acetyl-CoA Per 9glucose oxaloacetate

pyruvic acid 2 NADH 2 ATP 2 NADH 2 ATP 2 NADH 2 NADH citric acid 2 FADH2 oxaloacetic acid 2 CO2 2 CO2 Krebs cycle 2 CO isocitric acid 2 malic acid Note label is in OA after one turn of cycle, half the time fumaric acid on top, half on bottom. So α keto glutaric acid no CO2 from Ac-CoA after succinic just one turn. (CO2 in first acid turn is from OA). Succinic 9 Per glucose10 2 NADH 2 ATP pyruvic acid 2 NADH 2ATP 2 NADH 2 NADH

2 FADH2 citric acid 2 NADH oxaloacetic acid 2 CO2 2 CO2 Krebs Cycle 2 CO isocitric acid 2 malic acid

fumaric acid α keto glutaric acid

succinic acid 10 Glucose + 6 O2  6 CO2 + 6 H2O :

By glycolysis plus one turn of the Krebs Cycle:

1 glucose (6C)  2 pyruvate (3C)  6 CO2 √

2 X 5 NADH2 and 2 X 1 FADH2 produced per glucose 4 ATPs per glucose (2 from GLYCOL., 2 from KC as GTP)

NADH2 and FADH2 still must be reoxidized …. No oxygen yet to be consumed No produced yet

Paltry increase in ATP so far 11 Oxidation of NADH by O2

NADH2 + 1/2 O2 --> NAD + H2O ΔGo = -53 kcal/mole

If directly coupled to ADP  ATP (7 kcal cost), 46 kcal/mole waste, and heat

So the electrons on NADH (and FADH2) are not passed directly to oxygen, but to intermediate carriers,

Each transfer step involves a smaller packet of free negative energy change (release) 12 The (ETC)

Iron-sulfur NADH2 NAD FMNH2 CoQ FADH2 The electron transport chain +3 NADH Fe FMN FAD CoQH2 Net: NADH2 + ½ O2  NAD + H2O Fe+2 Cytb-Fe+3

CoQ +2 +2 Fe Cytb-Fe Cytc1-Fe+3 Fe+3 +2 Cytc-Fe energy Free ~ Cytc1-Fe+2 Cyta-Fe+3 Cytc-Fe+3 +2 Cyta-Fe+2 Cyta3-Fe + Cyta3-Fe+3 O2 + 4H heme 2H2O

Ubiquinone, or Coenzyme Q are Up to 50 C’s long 13 H FMN FMNH2

2H+, 2e-

R R H Ubiquinone, or heme Coenzyme Q

heme Up to 50 C’s long a protein with a heme in a pocket 14 Mitochondria and the chemiosmotic theory of oxidative

Electron transport chain (ETC) is in the inner membrane. Oxidative phosphorylation is in the lollipops (separate from ETC). The Krebs cycle are inside the mitochondria, in the matrix. The enzymes of glycolysis are outside the mitochondria, in the cytoplasm. 15 The electron transport chain H+ (protons) are pumped out as the electrons are transferred

(outside)

& FADH2 FAD

Complex: I II * III IV

Nelson and Cox, Principles of *https://en.wikipedia.org/wiki/Succinate_dehydrogenase 16 Schematic idea of H+ being pumped out

Inter-membrane Compartment +

+ e- + e-

+

Conformational Relaxation change back (absorbs energy) 17 FoF1 Complex: Oxidative phosphorylation (ATP formation)

18 Close-up of , showing proton flow

ATP synthase ? the inside ? crista + ADP + + + + + ATP + + the outside

What about E. coli? Its membrane houses all components 19 Chemiosmotic theory

Proton motive force (pmf) Chemical gradient Electrical gradient Water-pump-dam analogy Peter Mitchell 1961 (without knowing mechanism) “The Mitchell Hypothesis” Nobel Prize 1978

20 A test of the chemiosmotic theory Artificial phospholipid membrane

H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+

H+ H+ H+ H+

ETC Complex I’s

+NADH H+ H+ H+ Add NADH H+H+ H+ H+ H+ H+ H+ H+ H+ pH drops

21 A second test of the chemiosmotic theory

H+ H+ + + + H+ H H H + + + H+ H+ H H H H+ H+ H+ H+ + H+ + + H H+ H H + + + H H+ H H + H H+ + H + H+ ADP+Pi H H+ + H + H H+ H+ H+ H+

+ + H H H+ + + H H H+

22 H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ ADP + Pi H+ H+ H+ ATP H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ Artificially produced mitochondrial membrane vesicle with ADP and Pi trapped inside 23 ATP is formed from ADP + Pi A third test: Dinitrophenol (DNP): an uncoupler of oxidative phosphorylation

 - + H+

DNP’s -OH is weakly acidic in this environment. DNP can easily permeate the mitochondrial inner membrane.

Outside the , where the H+ concentration is high, DNP picks up a proton.

After diffusing inside, where the H+ concentration low, it gives up the proton. So it ferries protons from regions of high concentration to regions of low concentration, thus destroying the proton gradient. Electron transport chain goes merrily on and on, but no gradient is formed and no ATP is produced.

24 Next: How is the ATP actually made (from ADP + Pi) by simply re-entering the mitchochondrion?

25 ATP synthase (or the F0F1 complex) aka the lollipop the inside outside

ɣ

the outside inside Gamma subunit: cam ATP synthase Gamma subunit is inserted inside 26 not top view ATP synthase β fixed fixed α α inside β β

α http://employees.csbsju.edu/hjakubowski/classe s/ch331/oxphos/olcouplingoxphos.html a subunit c subunit fixed

not outside fixed

Flow of protons turns the C-subunit wheel. C-subunits turn the gamma cam. 27 View of c-subunits using atomic force microscopy

Norbert Dencher Animation of the F0 rotation driven and by the influx of H+ ions (“wheels within wheels”). Andreas Engel M.E. Girvin 28 Movie link 29 ATP synthase action

Start here (top view) Alpha+beta ADP Pi + + +H +H +H+ Gamma

Three conformational states of the α-β subunit: L, T, and O 30 Outside

Mitochondria

Inside 31 Is it really a motor?

Actin molecules Attach a big arm

Detach the C-subunits

10 nm 10 total length =~1 micron

32 Testing the ATP synthase motor model by running it in reverse (no H+ gradient, add ATP) actin filament Actin labeled Actin is a muscle by tagging it with protein polymer fluorescent molecules Attached to the gamma subunit

Add lots of ATP His-tag

Hiroyuki Noji, Ryohei Yasuda, Masasuke Yoshida & Kazuhiko Kinosita Jr. (1997) Direct observation of the rotation of F1-ATPase. Nature 386, 299 - 302. 33 The arm can be seen!

http://www.colum bia.edu/cu/biology/ courses/c2005/mo vies/fof1_rot_2700 nm.mpg 34 Run reaction in reverse: add ATP  drive counter-clockwise rotation of cam Here the driving motor (c) has been cut away from the cam (γ)

4 3 2 1 5

ATP

       

Start here

counter-clockwise Blue subunit= gamma subunit, cam rotation driven by αβ subunits. Notice the cam is driven to rotate counterclockwise 35 ATP synthase (F0F1) Some numbers:

MW = ~500,000 Use less load faster. Extrapolate to zero load: 6000 rpm! 100 rps so est. 300 ATP/sec. Per FoF1 molecule. ~100 trillion cells per person ~~1000 mitochondria per human cell. Each one riddled with FoF1 ATP synthase molecules. All spinning at 6000 rpm . . . . Picture it.

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