LECTURE 13 - ETC AND OXPHOS II

Question 1

what is evidence for the fact that the ETC is based on chemi-osmotic coupling?

Answer 1

1. the respiratory chain can function without phosphate
2. the number of ATP generated by NADH oxidation is not an integer
3. an intact IMM is required for OXPHOS
   4.key electron transport proteins span the IMM
4. uncouplers such as (2,4-nitrophenol) DNP inhibit ATP synthesis (weak acids that can take protons, go through the membrane and release the protons in the matrix)
5. generating an artificial proton gradient permits ATP synthesis without electron transport

Question 2

how do mitochondrial poisons work?

Answer 2

by blocking specific complexes and using artificial electron donors, you can find out exactly how much energy each complex contributes to the membrane potential

Question 3

what was the experiment that determine the contribution of complex I to the membrane potential

Answer 3

1. blocked complex III by using antimycin A which stops electron flow beyond complex 3
2. added ferricyanide (artificial electron donor)
3. added beta-hydroxybutyrate (feeds electrons into complex 1 by generating NADH)
4. under these conditions, the P/O ratio was 1, which confirms that complex 4 pumps 4 protons

Question 4

what does the P/O ratio mean?

Answer 4

ATPs produced per 1 oxygen atom

Question 5

what was the experiment to isolate complex IV?

Answer 5

1. Added ascorbate and TMPD (donate electrons directly to cytochrome C and skip earlier complexes)
2. With only complex 4 contribution, P/O ratio was 1
3. Complex IV pumps 2 protons per oxygen molecule, so why was the ratio 1 instead of 0.5?
4. Could be because 2 protons are used to reduce oxygen to water and 2 contribute to proton pumping but we are not sure

Question 6

what was the experiment to isolate complex III?

Answer 6

1. Added exogenous cytochrome C (to extract electrons directly from complex 3)
2. Added cyanide to inhibit complex 4
3. Added succinate (electron donor that directly donates complex 3 and skips complex 1)
4. With only complex 3 contribution, P/O ratio was 0.5

Question 7

what do uncouplers do?

Answer 7

Uncouplers work by dissipating the proton gradient across the mitochondrial membrane, preventing ATP synthesis despite continued electron flow and oxygen consumption

Question 8

how does dinitrophenol (DNP) work?

Answer 8

1. weak acid that can shuttle protons across the IMM
2. picks up protons from the intermembrane space and then releases them in the matrix, dissipating proton gradient without it passing through ATP synthase
3. this disrupts ATP production because the proton motive force needed to drive ATP synthesis is no longer there
4. In the 1930s, DNP was used for weight loss due to its ability to increase energy expenditure by uncoupling the ETC
   However, DNP has very narrow therapeutic window

Question 9

how does uncoupling protein 1 work (UCP1)?

Answer 9

1. Endogenous uncoupler (found in body)
2. mitochondrial protein that allows protons to re-enter the matrix without producing ATP
3. increases energy expenditure

Question 10

what is the size of the mitochondrial genome?

Answer 10

16.5 kB

Question 11

how many proteins does mitochondrial DNA encode?

Answer 11

13 proteins, all of which are essential for the ETC

Question 12

where are most mitochondrial proteins encoded?

Answer 12

mitochondria need almost 2000 proteins to function
most are encoded by nuclear DNA
they are then imported into the mitochondria
some of them are the ones needed by the mitochondria to do transcription and translation

Question 13

what can mutations in mitochondria DNA lead to?

Answer 13

lead to mitochondrial diseases, which are metabolic disorders:
1. muscle function
2. mental health
3. systemic issues

Question 14

how come some mitochondrial diseases do not originate in mitochondrial DNA?

Answer 14

mitochondrial biology involves coordination between nuclear and mitochondrial genome (for example complex I)
therefore, there are
1. Diseases caused by mutations in nuclear-encoded genes.
2. Disorders related to the assembly factors required for proper mitochondrial function.

Question 15

what is respiratory control?

Answer 15

Regulation of electron flow in the electron transport chain (ETC) depending on availability of:
oxygen
substrate (NADH)
ATP levels

Question 16

what is the role of ATP in respiratory control?

Answer 16

if ATP is high and ADP low
electrons flow and oxygen consumption will stop because the proton gradient is not being dissipated
Electron flow will resume when ATP gets consumed and converted back into ADP which triggers the proton flow through ATP synthase and pulls the electrons through the ETC

Question 17

how was oxygen consumption experimentally analysed?

Answer 17

researchers quantified the rate at which isolated mitochondria consume oxygen using oxygen electrode
oxygen electrode: device placed in a chamber containing isolated mitochondria and measures the concentration of oxygen
added glutamate: caused a slight decrease in oxygen levels
added ADP and Pi: caused a steeper decline in oxygen levels

Question 18

how did oxygen consumption change when DNP was added?

Answer 18

Glutamate: Introduced initially, resulting in a small decrease in oxygen levels due to substrate oxidation
ADP: Added next, significantly increases oxygen consumption as ATP synthesis occurs
DNP: Introduced later to demonstrate the uncoupling effect
-> the DNP uncoupler increases O2 consumption but not ATP synthesis, it uncoupled the two processes