Ox-Phos

Front 1

What does the chemiosmotic hypothesis propose?

Describe the experiment that suggested this.

Back 1

You can couple translocation of protons down a gradient with ATP synthesis.

Made lipid vesciles (by sonicating lipid bilayers) and had BACTERIORHODOPSIN (takes energy from light to make a proton gradient) and hat mitochondrial ATP synthase.

When light shone on it, protons pumped into vesicle and would flow out and make ATP.

Front 2

How does the mitochondrial ATPase make ATP?

Why was it originally called ATPase?

Back 2

Protons flow down their gradient thorugh ATP synthase to create ATP.

In original experiment, activity discovered was ATP hydrolysis-->Hydrogen being pumped against gradient.

But in cell, proton gradient is much bigger than the energy of ATP, so reverse happens.

Front 3

Describe the F1F0 composition of ATPase.

Back 3

F0 is in membrane, involved in proton translocation, has 10 c proteins that form a c ring, and 1 a protein.

F1 is cytosolic side (ATP synthase) and has 6 alpha subunits, 3 beta which are identical and a central gamma

Gamma interacts with c ring and beta.

Front 4

If the beta subunits of F1F0ATPase are identical why do they have different functions?

Back 4

They surround the gamma subunit, which is not symmetrical, so they all have different quaternary structure and thus different protein activities.

Front 5

If you hydrolyzed ATP with O18 water what would you get?

Would this occur in the absence of a proton gradient?

Back 5

ADP and 18O labeled Pi.

Well, the three beta units of ATPase have different functions: O, L, T
O loosely binds ADP + Pi
L has ADP and Pi stuck
T has ADP + Pi <--> ATP in equilibrium, but can isolate and push towards ATP synthesis).
Gamma turns CCW 120 degrees and then O becomes L, T becomes O, and L becomes T, resulting in release of ATP and binding of ADP and Pi.

SO you can get ATP formation and hydrolysis, but it's never being released! This is only turning when have proton gradients.

Front 6

How was it experimentally shown that there was ATP-driven rotation in ATP synthase?

Back 6

Attached fluorescently labeled actin filament to gamma unit and upon ATP hydrolysis, span CCW.

Front 7

Describe how the subunit composition of F0 contributes to rotation of gamma.

Back 7

Have c ring made of 10 c subunits having aspartic acid in center and have a subunit that has hald channels, one faces cytosol (lots H+) and other faces matrix (little H+).

C-ring can only turn if protonated (loses charge can interact and move through lipid bilayer). So H+ comes through cytosolic facing channel and protonates c subunit and it moves clockwise, then it dumps his proton in the matrix.

Front 8

How does ATP get out of the mitochondria? What are the two driving factors of the process?

Back 8

ATP-ADP Translocase: When open to cytosolic side has affinity for ADP so it binds it, undergoes eversion, faces matrix, less affinity, disassociates into matrix, binds ATP, eversion, loses ATP.

High concentrations of ATP in the matrix.

Voltage potential (inside is negative, ATP is more negative than ATP).

Front 9

What is decoupling? Effects of DNP?

Back 9

Decoupling is process of creating heat (not ATP) when hydrogen enters via gradient. This keeps you warm.
ATP synthesis inhibited.
Fat is burned up to compensate for inefficiency of ATP synthesis.

DNP has pka such that is is protonated in cytosol, passes through into innermembrane and releases proton, causing increase in bodily temperature, and protein breakdown.