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 ∆Go is substantial and negative.
1 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 ∆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 glycolysis: acetyl-CoA 3 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 α keto glutaric acid
succinic Handout 9A acid 3 Acetyl-OCoA O||
CH3 –C –OH + co-enzyme A acetyl~CoA acetic acid (acetate)
coA acetate group
pantothenic acid (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
succinic 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 dehydrogenase 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 water produced yet
Paltry increase in ATP so far Hans Krebs 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 electron transport chain (ETC)
Iron-sulfur protein 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 Cytochromes are proteins 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 cytochrome protein with a heme in a pocket 14 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. 15 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 *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 crista, showing proton flow
ATP synthase ? the inside ? crista + ADP + + + + + ATP + + the outside
What about E. coli? Its cell membrane houses all components 19 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
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 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.
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 hydrolysis
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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