Chemiosmosis, ATP synthesis, mitochondria | Quizlet

How does chemiosmosis allow ATP synthesis?

Electron transport gives energy to set up a proton gradient (gives proton-motive force).
The energy of protons flowing down the gradient drives synthesis of ATP.

Experiment to prove that just diff in conc. doesnt affect movement if no diff. in charge

Initially:
ph = 9 of buffer
"matrix" filled with KCL (net neutral charge) and 10^-9 conc. of H+
&
"IMS" filled with that same conc. of H+

--> so charges are equal


Then:
ph = 7 in new buffer, IMS also now has that higher H conc. since outer membrane is porous to buffer surroundings

HOWEVER, ph=7 so equal concentration of H+ and OH-, meaning no net charge!
No ATP synthesis happens since theres only a conc diff and not a charge diff in H+

But if we add Valinomycin (K+ passive gradient carrier) it will carry K+ into IMS from matrix due to lack of conc. in IMS -> THIS introduces a charge difference that allows ATP synthase to work

ATP synthase structure

F0 unit embedded in inner membrane, F1 unit on mitochondrial matrix side

F0: proton transport occurs from IMS to matrix (partially hydropobic since embedded to membrane)
- turns in its favorable direction as it picks up the proton and rotates

F1: site of ATP hydrolysis -> three aB dimer subunits
- forced to rotate in unfavorable dir, doing the also unfavorable rxn of ADP -> ATP



-> F0 and F1 are linked by a subunit so they must rotate in same direction
- at each point in time one of them rotates in preferred direction, other is unpreferred

V-ATPase (found in lysosomes, for example)

The F1 atpase does its favorable rxn instead and hydrolyzes ATP-> ADP. Then, it uses that energy to spin F0 in its unfavorable way and pump H+ into the lysosome FROM the cytosol.

used when organelles need high acidity)

F1 open vs loose vs tight

F1 exists in three aB dimers, each of which can exist in three diff confs:

open: empty
loose: binding ADP and Pi
tight: catalyzes ATP formation

- at any given time, one dimer will be loose, another open, and another tight (all confs are taken at all times)
- each conf change requires input of 3H+ from P (IMS) to N (matrix) side
- so for EACH subunit, need 9 h+ moved to get from open -> tight, but at any given time at least one of the dimers will be synthesizign ATP

-> 3 ATP molecules per translocation of 9 protons
-> = 1 ATP per 3 protons

How to prove rotation of C ring (F0 unit)

Attach actin with biotin (streptavidin) to watch the filament- we see that it rotates

Why is it 2.5 and 1.5 ATP for NADH and FADH2 (not 3 and 2 as expected from pumping 10H+ and 6H+)?

1) protons are used to facilitate transport processes
- to make ATP, we need ADP and Pi in mitoch... how do we get it? :

- when ATP made and sent from matrix to IMS, bringing in an ADP from IMS to matrix (net -1 out to IMS which is favorable since matrix already so negative)
- but you also need to bring in a phosphate into the already negative matrix-> so couple it with a H+ (losing one proton)


2) coenzymes like coQ are leaky and some electrons can just leave, so less energy to pump protons

what happens to NADH produced through PDC or CAC?

these occur in mitochondria, so they just donate electrons to complex I and return to their cycles as NAD to pick up e-

But what happens to NADH or FADH2 produced in cytosol (from glycolysis)?

need a way to get it into the mitchondria:

1) malate-asparatate shuttle

2) G3P shuttle

Malate-asparatate shuttle

1) malate-asparatate shuttle
- malate dehydrogenase causes oxaloacetate -> malate (bc malate can be transported into mitoch but oxaloacetate cannot) while doing NADH->NAD+
- then malate goes into mitoch and the same enzyme turns it back to oxaloacetate while doing NAD -> NADH
(oxaloacetate is reduced version of malate)

- to transport oxaloacetate back out, we turn it to aspartate by aminating (often from glutamate, which turns to a-ketoglutarate after donating amino group)
- then it gets into cytosol and deaminated to oxaloacetate


- sending NADH into mitoch but with many rxns/intermediates

G3P shuttle

G3P oxidized to DHAP while FAD accepts electrons, turning to FADH2 in matrix (via mitoch part of G3PDH)

DHAP reduced to to G3P, NADH turns to NAD (via cytosolic/IMS part of G3PDH)

- so you are sending FADH2 into mitoch

How many ATP from the 2 NADH produced in glycolysis then?

Either 3 or 5 ATP

If using malate-asparate shuttle: 2.5 ATP x 2 NADH = 5

if using G3P shuttle: 1.5 ATP x 2 FADH2 = 3 ATP

theoretical vs actual yield of ATPs from glucose? (using the 2/3 ATP vs 1.5/2 ATP numbers)

38 ATPs - theoretical

30 or 32 ATPs - actual

UCP-1 uncoupling protein (for babies and hibernating animals)

- just a passive channel allowing H+ to go through it in a rather uncontrolled way, from P side to N side (into matrix)
- this is favorable, but since energy doesnt need to be coupled to any rxn it gets used to generate heat

DNP and FCCP uncouplers

Have proton bound and unbound state, hydrophobic

- pick proton on one side, transport across membrane, drop it off

3 ways to inhibit oxidative phosphorylation

- inhibitors of ETC (rotenone for C1, antimycin A for C3)

- uncouplers (DNP proton carrier that blocks ATP synthesis, UCP1)

- terminal ATPase inhibitors (oligomycin)

What pH change will you see after you add mitcoh, succinate, and O2

It will drop once adding O2 since with this final carrier, proton pump can function to send H+ into IMS (until you run out of O2)

What pH change in IMS if add a mitoch and just ATP

a decrease after adding ATP since ATPase will use energy from ATP hydrolysis -> ADP to pump protons into IMS

What pH change if add mitoch and then DNP, then ATP and succinate?

NO ph change bc you added the DNP coupler, which will dissipate the gradient as it is made by the ETC

How does oligomycin block ATP synthesis

blocks ATPase

How does atractyloside block ATP synthesis

inhibits ATP/ADP antiporter, so no materials for making ATP

How will FCCP reverse impact of oligomycin? what O2 consumption change will be seen?

It allows dissipation of the proton gradient, allowing there to be no more buildup of protons in the IMS and ETC to function to send more protons into the IMS

can now consume O2 as ETC can function

How do mitochondria get degraded?

They get marked for microphagy when they get depolarized (when the matrix fills with H+, signaling that ATP synthesis is not working well)

When mitochondria don't work and their proteins (such as cytochrome C) leak into cytosol, what happens?

trigger cell death

cyt C release triggers cell death by activating apoptasome, which activates caspases
- no ATP so no more scramblase/flippase/floppase so phopshtidylserine conc increases on outer membrane, marking cells for death by engulfatin by other cells

Why is mitochondrial DNA so important?

it encodes essential proteins for ETC as well as ATPase

ROS in mitochondria

- Reactive oxygen species- produced during ETC, coenzyme Q has high affinity for O2 in addition to substrate CIII -> so it can make oxygen superoxide radicals (single e- transfers)

- This is why mitoch. need high level of NADPH which can turn GSSG to GSH which can turn H2O2 to H2O

- NADPH is made in mitoch simply by pulling H from NADH and putting it on NADP+ via hydride exhange complex

How and why is NADPH made in mitoch

- NADPH is made in mitoch simply by pulling H from NADH and putting it on NADP+ via hydride exhange complex

- mitoch. need high level of NADPH which can turn GSSG to GSH which can turn H2O2 to H2O -> bc of superoxide radicals, ROS

can there be diverse mitochondria within organisms

yes each cell can have diff mutated mitoch - hetereoplasmy (not all mitoch have same DNA)

Mutations in mitoch genome lead to ____

complex physiological disorders

(ex. liver failure, nerve damage, diabetes)

ex. diabetes - need energy to make vesicles to secrete insulin out as a sign of high blood sugar- without mitoch, no ATP for insulin secretion

How do protons get from one side of membrane to other?

Amino acids in membrane called “proton wires”