Intro Bio Lecture 9
1 Characteristics of metabolic pathways
Aside from its role in energy metabolism:
Glycolysis is a good example of a metabolic pathway.
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- glucose-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
citric acid oxaloacetic acid Krebs cycle Citric acid cycle isocitric acid malic acid Tricarboxylic acid cycle TCA cycle
fumaric acid 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
succinic 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 mitochondrion, 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 + 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 57 Fatty acido -2 Acetyl-CoA 58