Oxidative Phosphorylation - RS Flashcards
When is a natural uncoupler typically activated?
It is activated to ‘short-circuit’ mitochondrial energy coupling for the purpose of producing heat (thermogenesis)
When is a natural inhibitor protein used?
It is used to inactivate the ATP synthase under ischemic conditions where ATP synthase would act to rapidly hydrolyze cellular ATP.
What do uncouplers do?
They uncouple oxidation from phosphorylation. They stimulate oxygen consumption in the absence of ADP. Uncouplers dissipate the proton gradient by providing an alternate route for proton re-entry into the matrix. Acid + Base = Water and heat. (In the absence of a gradient, the synthase runs in reverse hydrolyzing ATP.- this fact is more important for inhibitors)
What are properties of a good uncoupler? What’s an example of one?
They are weak acids, hydrophobic, and have delocalized charge. Dinitrophenol is a classic uncoupler.
How does “the uncoupler protein” work?
It is a proton pore which is under careful hormonal regulation. Norepi binds, adenylate cyclase is activated, PKA is activated, free fatty acids are released by triglycerol lipase and they bind to the uncoupler protein on the mitochondria opening the channel.
What is a phosphorylation inhibitor? What is its purpose?
It prevents ATP-synthase from working. A natrual phosphorylation inhibitor (the inhibitor protein) protects against ATP hydrolysis during ischemia. It binds when the cell is absent of O2, pH drops and the inhibitor protein is protonated and binds to FoF1. When O2 is reintroduced protons are pumped out of the matrix, the inhibitor is deprotonated and ATP synthesis resumes. Prevents ATP from being hydrolyzed to re-establish the H+ gradient.
What is the classic example of a phosphorylation inhibitor?
Oligomycin.
From and Oxygen consumption measurement what happens when Oligomycin is introduced to a cell?
Oligomycin blocks proton movement through FoF1. Respiration slows because protons no longer reenter the matrix through FoF1. Uncouplers stimulate respiration, but no ATP is made. It blocks ADP- but not uncoupler stimulated respiration
Introducing cyanide into a cell has what effect on ADP and uncoupler stimulated respiration?
They are both blocked by CN-. The inhibitor protein will bind to the ATP synthase. CN competes for heme a3 and CuB.
Where does Rotenone effect the respiratory chain?
In the beginning (amytal does too). It is found in barbituates
Where does Antimycin A block the respiratory chain?
In the middle. (just before cytochrome C1 and Cyt C)
What is the chemiosmotic theory?
A delocalized electrochemical gradient is a required intermediate in coupling the exergonic redox reactions to the endergonic synthesis of ATP (similar to common intermediate principal). Mechanism Mitchell proposed is that membranes are impermeable to protons and an electrochemical gradient exists.
What is Mitchell’s loops? What is the result?
The overall principle is that protons are transported as a direct result of the chemistry of the oxidation-reduction reactions combined with the vectorial nature of the sequence. A H+ acceptor (Q) is reduced on the inside surface of the IMM by e- donors. Two protons are extracted from the matrix. The Q is then moved to the outer surface of the IMM. 1 e- is passed down to oxygen and the other e- is recycled, but two protons are transported. Thus the stoichiometry is 2H+/ e-
Why can’t Mitchell’s proposed Loops function in cytochrome oxidase?
There is no H donor/ acceptor. An indirect coupling mechanism is used instead. Protein conformation changes are utilized.
What is the cost of having cytoplasmic NADH vs matrix NADH?
There is less ATP yielded from NADH on the cytoplasmic side.
