Intro Bio Lecture 9

1 Characteristics of metabolic pathways

Aside from its role in energy metabolism:

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 DGo is substantial and negative.

2 dihydroxyacetone phosphate

fructose-1,6- -6- fructose-6- diphosphate phosphate phosphate glyceraldehyde-3-phosphate Glycolytic pathway, Glycolysis glucose 1,3-diphosphoglyceric acid

phosphoenol-pyruvic 2-phosphoglyceric 3-phosphoglyceric acid acid acid

yeast lactic acetaldehyde ethanol acid pyruvic acid oxidative pathways Handout 8A 3 Fermentation goes all the way to the right Efficiency: glucose--> 2 lactates, without considering the couplings for the formation of ATP's (no energy harnessing): Δ Go = -45 kcal/mole kcal/mole Out of this comes 2 ATPs, worth 14 kcal/mol. 31 kcal/mole output as heat. So the efficiency is about 14/45 = ~30% Not bad at all.

Since 2 ATPs ARE produced, taking them into account, for the reaction: Glucose + 2 ADP + 2 Pi → 2 lactate + 2 ATP ΔGo = -31 kcal/mole (45-14) Very favorable. All the way to the right. Keep bringing in glucose, keep spewing out lactate, Make all the ATP you want. 4 Energy yield But all this spewing of lactate turns out to be wasteful.

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

How much energy released then?

Glucose + 6 O2 → 6 CO2 + 6 H2O DGo = -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 5 From glycolysis: acetyl-CoA 6 pyruvic acid Scores: per glucose 2 NADH 2 ATP pyruvic acid 2 NADH

2 CO2

Krebs cycle Tricarboxylic acid cycle TCA cycle

a keto glutaric acid

succinic Handout 9A acid 6 Acetyl-OCoA O||

CH3 –C –OH + co-enzyme A → acetyl~CoA acetic acid (acetate)

coA acetate group

pantothenic acid (vitamin B5) 7 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 a keto glutaric acid

8 GTP is energetically equivalent to ATP

GTP + ADP → GDP + ATP

ΔGo = ~0

G= guanine (instead of adenine in ATP)

9 acetyl-CoA B Per 10glucose 2 oxaloacetate 2 NADH 2 ATP pyruvic acid 2 NADH 2 NADH 2 ATP 2 NADH 2 FADH2 citric acid 2 NADH oxaloacetic acid 2 CO2 2 CO2 Krebs cycle 2 CO2 isocitric acid 6 CO malic acid 2

fumaric acid a keto glutaric acid

succinic acid 10 FAD = flavin adenine dinucleotide

Business end (flavin) ~ Vitamin B2 ribose

adenine -

ribose FAD + 2H. → FADH 2 11 D acetyl-CoA Per 12glucose 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 a keto glutaric acid no CO2 from Ac-CoA after succinic just one turn. (CO2 in first acid turn is from OA). Succinic dehydrogenase 12 E

Per glucose13 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 a keto glutaric acid

succinic acid 13 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 water produced yet

Paltry increase in ATP so far Hans Krebs 14 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) 15 The electron transport chain (ETC)

Iron-sulfur protein NADH2 NAD FMNH2 CoQ FADH2 The electron transport chain NADH Fe+3 FMN CoQH FAD 2 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 Cytochromes are proteins Up to 50 C’s long 16 H FMN FMNH2

2H+, 2e-

R R H Ubiquinone, or heme Coenzyme Q

heme Up to 50 C’s long a cytochrome protein with a heme in a pocket 17 Mitochondria and the chemiosmotic theory of oxidative phosphorylation

Electron transport chain (ETC) is in the inner membrane. Oxidative phosphorylation is in the lollipops (separate from ETC). The Krebs cycle enzymes are inside the mitochondria, in the matrix. The enzymes of glycolysis are outside the mitochondria, in the cytoplasm. 18 The electron transport chain H+ ions (protons) are pumped out as the electrons are transferred

(outside)

FADH2→ FAD Complex: I II III IV

Nelson and Cox, Principles of Biochemistry 19 Schematic idea of H+ being pumped out

Inter-membrane Compartment +

+ e- + e-

+

Conformational Relaxation change back (absorbs energy) 20 Outline of Energy Metabolism

OXPHOS: 1 NADH from glycolysis Substrate level phosphorylation 1 NADH from Krebs pre-entry (SLP): 2 ATP total: 3 NADH from Krebs

1 ATP from 1 FADH2 from Krebs Glycolysis we recover NAD and FAD by 1 ATP (GTP) ETC (4), but from Krebs WHAT ABOUT ATP (5)?

21 FoF1 Complex: Oxidative phosphorylation (ATP formation)

22 Close-up of crista, showing proton flow

the inside 1 pair of NADH electrons going down ATP the ETC → ~10 H+’s pumped out synthase crista + ADP + + + ATP + + + + + + + + + the outside

23 Close-up of crista, showing proton flow

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

~10 H+’s ~10 H+’s 3 ATP 3 ADP + 3 Pi

1 NAD + 1 H2O 1 NADH2 + ½ O2 24 Close-up of crista, showing proton flow

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

What about E. coli? Its cell membrane houses all components 25 Chemiosmotic theory

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

26 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

27 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+

28 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 29 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.

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

31 ATP synthase (or the F0F1 complex) MW = ~500,000 the inside H+ outside a c

F0

e g b

b a F d 1

the outside inside Gamma subunit acts as a cam Gamma subunit is inserted inside the αβs 32 Cryo electron microscopy

Math, computational algorithms

Joachim Frank 2017 Nobel Prize in Chemistry* Rubinstein JL, Walker JE, Henderson R. Structure of the mitochondrial ATP synthase by *Dept. of Biological Sciences electron cryomicroscopy. The EMBO Journal. Columbia University ☺ 2003;22(23):6182-6192. 33 34 not top view ATP synthase fixed b fixed inside a a b b

a 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. 35 Movie link 36 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 37 ATP synthase action

38 Outside

Mitochondria

Inside 39 Is it really a motor?

Actin molecules Attach a big arm

Detach the C-subunits

10 nm

total length =~1 micron10

40 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

Junge et al. (1997) TIBS 22, 420-423

Hiroyuki Noji, Ryohei Yasuda, Masasuke Yoshida & Kazuhiko Kinosita Jr. (1997) Direct observation of the rotation of F1-ATPase. Nature 386, 299 - 302. 41 Run reaction in reverse: add ATP → drive counter-clockwise rotation of cam Here the driving motor (c) has been cut away from the cam (g)

4 3 2 1 5

ATP hydrolysis

       

Start here

counter-clockwise Blue subunit= gamma subunit, cam rotation driven by ab subunits. Notice the cam is driven to rotate counterclockwise 42 The arm can be seen!

Extrapolate to zero load: 6000 rpm! 100 rps so ~300 ATP/sec

http://www.colum bia.edu/cu/biology/ courses/c2005/mo vies/fof1_rot_2700 nm.mpg 43 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.

44 More numbers: ATP accounting

For each pair of electrons given up by NADH: • Each of the 3 ETC complex (I, III, IV) pumps enough H+ ions to allow the formation of 1 ATP. • So 3 ATPs per pair of electrons passing through the full ETC.

• So 3 ATPs per 1/2 O2 • So 3 ATPs per NADH2 • But only 2 ATPs per FADH2 (skips complex I)

45 Nelson and Cox, Principles of Biochemistry 46 More exergonic DGo for no fumarate+NADH2 succinate + NAD fumarate formation succinate + FAD with FADH2 than with NADH yes H2

fumarate + FADH2

) 2

ATP ) (relative to O to ) (relative

ATP mol

ATP generated by the

’ (kcal/ ’

° G Free energy change energy Free ATP synthetase is ATP D called is oxidative phosphorylation, or oxphos. 47 Outline of Energy Metabolism

OXPHOS: 1 NADH from glycolysis Substrate level phosphorylation 1 NADH from Krebs pre-entry (SLP): 2 ATP total: 3 NADH from Krebs

1 ATP from 1 FADH2 from Krebs Glycolysis Total: 17 ATP 1 ATP (GTP) 5 NADH = 15 ATP from Krebs 1 FADH2 = 2 ATP

Grand total (E. coli): 17 + 2 = 19 per ½ glucose or 38 per 1 glucose 48 Cellular location: eukaryotes

CYTOPLASM

MITOCHONDRIA

In bacteria, the ETC is in the plasma membrane.

49 ATP accounting

• 38 ATP/ glucose in E. coli

• 36 ATP/glucose in eukaryotes • Cost of bringing in the electrons from NADH from glycolysis into the mitochondrion = 1 ATP per electron pair • So costs 2 ATPs per glucose, subtract from 38 to get 36 net.

50 Efficiency

• 36 ATP/ glucose, worth -7 X 36 = -252 kcal/mole of glucose

o • DG for the overall reaction glucose + 6 O2→ 6 CO2 + 6 H2O: -686 kcal/ mole • Efficiency = 252/686 = 37%

• Once again, better than most gasoline engines.

• and Energy yield: 36 ATP/ glucose vs. 2 ATP/glucose in fermentation (yet fermentation works)

• So with or without oxygen, get energy from glucose 51 Alternative sources of carbon and energy

French fries = fat (oil) = triglyceride

52 (Triglyceride) Lipases (hydrolysis)

Glycolysis (at DHAP)

53 Glycerol as an alternative sole carbon and energy source for E. coli

ATP +NAD + NADH2

DHAP glycerol glycerol phosphate (dihydroxy acetone phosphate)

- O glycolysis 2 +O ? 2

CO2 + H2O

54 and ADP + Pi → ATP Glycerol + ATP → glycerol phosphate → DHAP NAD → NADH2

55 ATP +NAD + NADH2

DHAP glycerol glycerol phosphate (dihydroxy acetone phosphate)

glycolysis - O2 +O2

Glycerol cannot be fermented. CO2 + H2O E. coli CANNOT grow on glycerol in the absence of air These pathways are real, and they set the rules. 56 Stoichiometry of chemical reactions must be obeyed. No magic is involved. Fatty acid catabolism (oxidation)

O || CH3-(CH2)n-CH2-CH2-C-OH

ATP + -SH O || CH3-(CH2)n-CH2-CH2-C-CoA + HOH

FAD FADH2 FAD FADH2 O || CH3-(CH2)n-CH=CH-C-CoA etc.

+HOH

OH O | || CH3-(CH2)n-CH-CH2-C-CoA

NAD NADH2

O O || || CH3-(CH2)n-C-CH2-C-CoA

+ CoA Krebs Cycle O O || || CH3-(CH2)n-C-CoA + CH3-C-CoA 57 Fatty acido -2 Acetyl-CoA 58