The H+ (protons) are pumped out as the electrons are transferred

(outside)

FADH2 FAD Complex: I II III IV

Nelson and Cox, Principles of Biochemistry 1 Close-up of , 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

2 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 3 Close-up of crista, showing proton flow

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

What about E. coli? Its cell membrane houses all components 4 ATP synthase (or the F0F1 complex) MW = ~500,000 the inside H+ outside a c

F0

ε γ b

α β F δ 1

the outside inside Gamma subunit acts as a cam Gamma subunit is inserted inside the αβs 5 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. 6 7 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. 8 Movie link 9 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 10 Outside

Mitochondria

Inside 11 Is it really a motor?

Actin molecules Attach a big arm

Detach the C-subunits

10 nm 10 total length =~1 micron

12 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 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. 13 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 hydrolysis

       

Start here

counter-clockwise Blue subunit= gamma subunit, cam rotation driven by αβ subunits. Notice the cam is driven to rotate counterclockwise 14 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 15 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)

16 Nelson and Cox, Principles of Biochemistry 17 More exergonic ∆Go for no fumarate+NADH2 succinate + NAD fumarate formation succinate + FAD with FADH2 than with NADH yes H2 fumarate + FADH2 ) 2 ATP ) O (relative to ATP mol

ATP generated by the ’ (kcal/ ° G Free energy change energy Free

ATP synthetase is ATP ∆ called is oxidative phosphorylation, or oxphos. 18 Outline of Energy Metabolism

OXPHOS: 1 NADH from 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 19 Cellular location:

CYTOPLASM

MITOCHONDRIA

In bacteria, the ETC is in the plasma membrane.

20 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 = 1 ATP per electron pair • So costs 2 ATPs per glucose, subtract from 38 to get 36 net.

21 Efficiency

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

o • ∆G 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 , get energy from glucose 22 Alternative sources of carbon and energy

Shake = milk: milk sugar = lactose = disaccharide = glucose – galactose

beta-galactosidase +HOH → glucose + galactose

glucose → glycolysis, etc.

galactose  (3 enzymatic steps)  glucose 23 Alternative sources of carbon and energy

Bun = starch = poly-alpha-glucose

G-1-P → G-6-P

glycolysis

24 Alternative sources of carbon and energy

Lettuce = cellulose = polysaccharide Poly-beta glucose

→| (stays as the polysaccharide)

We have no for catabolizing cellulose

25 Alternative sources of carbon and energy

French fries = fat (oil) = triglyceride

26 (Triglyceride) Lipases (hydrolysis)

Glycolysis (at DHAP)

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

ATP +NAD + NADH2

DHAP glycerol glycerol (dihydroxy acetone phosphate)

- O glycolysis 2 +O ? 2

CO2 + H2O

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

29 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. 30 Stoichiometry of chemical reactions must be obeyed. No magic is involved. Fatty acid catabolism (oxidation)

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

ATP + Coenzyme A-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 31 Fatty acido -2 Acetyl-CoA Alternative sources of carbon and energy

Hamburger = protein Proteases (e.g., trypsin) → → 20 AAs Stomach acid (pH1) also helps by denaturing protein making it accessable to proteolytic attack

Each of the 20 AA’s has its own catabolic pathway, and ends up in the glycolytic or Krebs cycle pathways

But first, the N must be removed:

32 Handout 9-2

Deamination and transamination of amino acids

Transamination

Alanine alpha-keto-glutaric acid Pyruvate Glutamic acid

Oxidative de-amination

HOH

NAD NADH 2 Glutamic acid alpha-keto-glutaric acid 33 E.g., degradation of phenylalanine (6 steps)

Phe builds up and gets metabolized to an injurious product (phenyl pyruvate)

PKU (phenylketonuria)

transaminase

Products = Fumaric acid → Krebs and Acetoacetate → 2 Acetyl-CoA → Krebs 34 You are what you eat Catabolism Anabolism

ATP STARCH glucose GLYCOGEN GLYCOLYSIS ATP pyruvate ATP FATS acetyl-CoA FATS

KREBS O.A. -K.G. AMINO ACIDS ATP ATP AMINO ACIDS

E.T.C. NADH 2 NAD ATP PROTEINS

O2 H2O

35 Handout 9-2 Flow of glucose in E. coli

Macromolecules Polysaccharides Lipids Nucleic Acids Proteins monomers

y a w th a p c ti e th intermediates n y s io b

glucose

36 Each arrow = a specific Handout 9-2

You are what you eat

Catabolism Anabolism

ATP STARCH glucose GLYCOGEN GLYCOLYSIS ATP pyruvate ATP FATS acetyl-CoA FATS

KREBS O.A. -K. G. AMINO ACIDS ATP ATP AMINO ACIDS

E.T.C. NADH2 NAD PROTEINS ATP PROTEINS

O 2 H2O 37 Handout 9-2 Biosynthesis of monomers

E.g., • Fatty acids (acetyl CoA from Krebs cycle) • Amino acids (Serine: 3-phospho-glyceric acid from glycolysis)

38 Fatty acid biosynthesis O || CH3-(CH2)n-CH2-CH2-C-OH Repeated addition of 2-carbon units (acetyl-CoA molecules via this same set of reactions) O || Handout 9-1 CH3-CH22 -CH -C-CoA

NADPH NADP Start at bottom 2 O || CH3-CH= CH-C-CoA

-HOH

OH O | || CH3-C-CH -C-CoA H 2

NADPH2 NADP O O || || + CoA CH3-C-CH2-C-CoA Krebs Cycle O O || || CH -C-CoA + CH -C-CoA 3 3 39 Acetyl-CoA Acetyl-CoA Handout 9-1 right Handout 9-3

Phosphoester group

(Glycolytic intermediate) Glutamate is the amino donor

hydrolysis

Handout 9-3 40 41 Biosynthesis of macromolecules

Flow of glucose in E. coli

Macromolecules Polysaccharides Lipids Nucleic Acids Proteins monomers

y a w th a p c ti e th intermediates n y s io b

glucose

42 Each arrow = a specific chemical reaction Biosynthesis of macromolecules

1) Lipids 2) Polysaccharides 3) Proteins

43 Triglyceride (fat) biosynthesis

DHAP NADPH (dihydroxy- Glycerol phosphate acetone phosphate)

O + Fatty acid 1 || -C-(CH ) -CH3 O 2 x || -C-(CH ) -CH3 O 2 x || -C-(CH ) -CH3 + Fatty acid 2 2 y

Phosphatidic acid

O + Fatty acid 3 || -C-(CH ) -CH3 2 x O || -C-(CH2)y-CH3 Phospholipid O || -C-(CH ) -CH3 2 z Triglyceride 44 Handout 9-3 45 Polysaccharide: Hyaluronic acid

n

46 Polysaccharide synthesis Hyaluronic acid (joint lubricant) N-acetyl-glucosamine Glucuronic acid

COO-

Enz.1 Enz. 2 Enz.1 + Enz. 2 Enz.1 Enz. 2 Enz.1 Enz. 2 Enz.1 Enz. 2 Enz.1

Hyaluronic acid (polysaccharide) via ~2 47 Biosynthesis of proteins e.g., an enzyme like hexokinase: met-val-his-leu-gly …..

If this done like lipids and polysaccharides, we need an enzyme for each linkage First an enzyme that will condense val to met to make met-val. Then an enzyme with a different substrate specificity, which adds his to met- val to make met-val-his. Since there are 500 AAs in hexokinase, we need 500 enzymes to do the job. If there are 3000 proteins in E. coli, then we need ~500 X 3000 = 1.5 million enzymes to make all the different primary structure of all the proteins. But even then, it won’t work, as each of these million enzymes is also a protein that needs to be synthesized. We need a better plan to polymerize the amino acids in the right order.

48 Flow of glucose in E. coli

Macromolecules Polysaccharides Lipids Nucleic Acids Proteins

= pre-existing stuff monomers

y a = newly synthesized stuff w th a p c ti e th intermediates n y s io b

Problem 1: Getting specific reaction rates to go in real time: glucose

Enzymes Each arrow = a specific chemical reaction

Problem 2: Getting the reactions to go in the desired direction: Coupled reactions + favorable metabolic paths (all mediated by enzymes)

Problem 3: Getting the information specific 3-D enzymes: Just need to specify the primary structure ….. How? 49