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BIOC1009 Dina > Experiment E: oxidative phosphorylation > Flashcards

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?

A

threeee

23
Q

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

A

two

24
Q

what are uncouplers?

A

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

25
Q

what is an example of an uncoupler?

A

2,4-dinitrophenol;

  • water-soluble when deprotonated
  • lipid-soluble when protonated
  • diffuses across the crista membrane down its concentration gradient
26
Q

what is the effect of adding an uncoupler?

A
  • 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
Q

what is the effect of adding an uncoupler in the presence of compounds that inhibit ATP-synthase or the transport of ADP or ATP?

A

rapid utilisation of oxygen as an uncoupler bypasses the ATP-synthase, negating the effect of the inhibitor

28
Q

in which situation will an uncoupler have no effect?

A

if the ETC itself is inhibited, since there is no proton gradient to be discharged by the uncoupler

29
Q

is ATP an exothermic or endothermic reaction?

A

endothermic; coupled with electron transport

30
Q

describe the mechanism of ATP-synthase

A

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
Q

what is the effect of protons entering the stalk of the primary particle?

A
  • cause a rotation of the central part of the ATP-synthase

- forcing each site to proceed to the next step in the reaction

32
Q

what class of inhibitor is dinitrophenol?

A

a respiratory stimulant

33
Q

what is the effect of dinitrophenol?

A
  • increased metabolic rate
  • high oxygen consumption
  • high body temperature
34
Q

which inhibitors act on complex I?

A

amytal and rotenone

35
Q

which inhibitors act on cytochrome c reductase?

A

antimycin A

36
Q

which inhibitors act on ATP synthase?

A

oligomycin

37
Q

which inhibitors act on cytochrome c oxidase?

A

potassium cyanide

38
Q

are you gona kill this exam tmro?

A

YES

39
Q

who is going to get 100% in this test?

A

I AM

BIOC1009 Dina (5 decks)

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