Experiment E: oxidative phosphorylation Flashcards

1
Q

how does the oxygen electrode measure the utilisation of oxygen?

A

by determining percentage oxygen saturation in the reaction mixture

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2
Q

how is the oxygen electrode calibrated?

A
  • calibration against a buffer saturated with oxygen to set 100% saturation
  • calibration against a buffer depleted of oxygen to set 0% saturation
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3
Q

what is the function of teflon?

A
  • non-stick frying pans
  • to separate the reaction mixture from the electrodes
  • it permits the diffusion of oxygen from the reaction buffer into the potassium chloride solution that bathes the electrodes
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4
Q

what are the electrodes made of?

A

platinum cathode and silver anode

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5
Q

how is the oxygen concentration determined?

A
  • a voltage is applied between the electrodes

- resulting current (~1μA) is proportional to the concentration of oxygen

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6
Q

which reaction occurs at the cathode?

A

reduction;

O2 + 4H+ + 4e- —> 2H2O

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7
Q

which reaction occurs at the anode?

A

oxidation;

4Ag + 4Cl- —> 4AgCl + 4e-

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8
Q

outline the experimental method

A
  • buffer and substrate allowed to equilibrate
  • mitochondrial preparation is added
  • consumption of oxygen measured
  • ADP/inhibitors/etc. are added
  • oxygen consumption measured until there is no further reaction
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9
Q

in the experiment, what was the amount of oxygen that corresponded to 100% oxygen saturation?

A

1327 nmol

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10
Q

which coenzymes react directly with substrates?

A

nicotinamide nucleotides

flavin coenzymes

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11
Q

which components of the ETC undergo redox reactions involving the transfer of both protons and electrons?

A
  • nicotinamide nucleotides
  • flavins
  • ubiquinone
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12
Q

which components of the ETC undergo redox reactions involving only electron transfer?

A
  • cytochromes

- non-haem iron proteins

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13
Q

what are the two types of nicotinamide nucleotides?

A
NAD = nicotinamide adenine dinucleotide
NADP = nicotinamide adenine dinucleotide phosphate
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14
Q

what is the reactive group of NAD and NADP?

A

the nicotinamide ring, which is positive when coenzymes are oxidised and neutral when it is reduced

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15
Q

what types of flavin coenzymes are there?

A
  • covalently-bound riboflavin
  • tightly- (non-covalently-) bound riboflavin monophosphate (also known as flavin mononucleotide, FMN) or flavin adenine dinucleotide (FAD)
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16
Q

how do flavin coenzymes undergo redox reactions?

A
  • as a single electron reaction

- or as two single-electron reactions by way of an intermediate flavin free radical

17
Q

how does ubiquinone undergo redox reactions?

A
  • in two single electron states

- via a half-reduced semiquinone radical

18
Q

what do cytochromes consist of?

A

a tetrapyrrole (porphyrin) ring chelating a central iron atom

19
Q

why are non-haem proteins also called iron-sulphur proteins?

A

iron atoms are chelated by sulphydryl group of cysteine residues and inorganic sulphide

20
Q

how do haems in different cytochromes differ?

A
  • in the substituents of the porphyrin ring
  • this affects their redox potential
  • permits different methods of binding to the proteins in different cytochromes
21
Q

what is the function of the electron carriers in the electron transport chain?

A
  • act to transfer the electrons derived from oxidation of substrates down an electrochemical gradient until oxygen is reduced to water
  • pump protons across the crista membrane to form a gradient with an excess of protons in the crista space
22
Q

at how many steps in the reoxidation of NADH is there phosphorylation of ADP to yield ATP?

23
Q

at how many steps in the reoxidation of reduced FAD is there phosphorylation of ADP to yield ATP?

24
Q

what are uncouplers?

A

weak acids that transport the protons back into the matrix directly, bypassing the ATP-synthase

25
what is an example of an uncoupler?
2,4-dinitrophenol; - water-soluble when deprotonated - lipid-soluble when protonated - diffuses across the crista membrane down its concentration gradient
26
what is the effect of adding an uncoupler?
- bypasses ATP-synthase - rate of oxidation is no longer controlled by the availability of ADP - complete utilisation of oxygen, regardless of the amount of ADP present
27
what is the effect of adding an uncoupler in the presence of compounds that inhibit ATP-synthase or the transport of ADP or ATP?
rapid utilisation of oxygen as an uncoupler bypasses the ATP-synthase, negating the effect of the inhibitor
28
in which situation will an uncoupler have no effect?
if the ETC itself is inhibited, since there is no proton gradient to be discharged by the uncoupler
29
is ATP an exothermic or endothermic reaction?
endothermic; coupled with electron transport
30
describe the mechanism of ATP-synthase
three equivalent catalytic sites at different stages of the reaction: - binding ADP and phosphate - catalysing the condensation of ADP and phosphate and releasing water - releasing ATP, ready to accept ADP and phosphate
31
what is the effect of protons entering the stalk of the primary particle?
- cause a rotation of the central part of the ATP-synthase | - forcing each site to proceed to the next step in the reaction
32
what class of inhibitor is dinitrophenol?
a respiratory stimulant
33
what is the effect of dinitrophenol?
- increased metabolic rate - high oxygen consumption - high body temperature
34
which inhibitors act on complex I?
amytal and rotenone
35
which inhibitors act on cytochrome c reductase?
antimycin A
36
which inhibitors act on ATP synthase?
oligomycin
37
which inhibitors act on cytochrome c oxidase?
potassium cyanide
38
are you gona kill this exam tmro?
YES
39
who is going to get 100% in this test?
I AM