Experiment E: oxidative phosphorylation Flashcards
how does the oxygen electrode measure the utilisation of oxygen?
by determining percentage oxygen saturation in the reaction mixture
how is the oxygen electrode calibrated?
- calibration against a buffer saturated with oxygen to set 100% saturation
- calibration against a buffer depleted of oxygen to set 0% saturation
what is the function of teflon?
- 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
what are the electrodes made of?
platinum cathode and silver anode
how is the oxygen concentration determined?
- a voltage is applied between the electrodes
- resulting current (~1μA) is proportional to the concentration of oxygen
which reaction occurs at the cathode?
reduction;
O2 + 4H+ + 4e- —> 2H2O
which reaction occurs at the anode?
oxidation;
4Ag + 4Cl- —> 4AgCl + 4e-
outline the experimental method
- 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
in the experiment, what was the amount of oxygen that corresponded to 100% oxygen saturation?
1327 nmol
which coenzymes react directly with substrates?
nicotinamide nucleotides
flavin coenzymes
which components of the ETC undergo redox reactions involving the transfer of both protons and electrons?
- nicotinamide nucleotides
- flavins
- ubiquinone
which components of the ETC undergo redox reactions involving only electron transfer?
- cytochromes
- non-haem iron proteins
what are the two types of nicotinamide nucleotides?
NAD = nicotinamide adenine dinucleotide NADP = nicotinamide adenine dinucleotide phosphate
what is the reactive group of NAD and NADP?
the nicotinamide ring, which is positive when coenzymes are oxidised and neutral when it is reduced
what types of flavin coenzymes are there?
- covalently-bound riboflavin
- tightly- (non-covalently-) bound riboflavin monophosphate (also known as flavin mononucleotide, FMN) or flavin adenine dinucleotide (FAD)
how do flavin coenzymes undergo redox reactions?
- as a single electron reaction
- or as two single-electron reactions by way of an intermediate flavin free radical
how does ubiquinone undergo redox reactions?
- in two single electron states
- via a half-reduced semiquinone radical
what do cytochromes consist of?
a tetrapyrrole (porphyrin) ring chelating a central iron atom
why are non-haem proteins also called iron-sulphur proteins?
iron atoms are chelated by sulphydryl group of cysteine residues and inorganic sulphide
how do haems in different cytochromes differ?
- in the substituents of the porphyrin ring
- this affects their redox potential
- permits different methods of binding to the proteins in different cytochromes
what is the function of the electron carriers in the electron transport chain?
- 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
at how many steps in the reoxidation of NADH is there phosphorylation of ADP to yield ATP?
threeee
at how many steps in the reoxidation of reduced FAD is there phosphorylation of ADP to yield ATP?
two
what are uncouplers?
weak acids that transport the protons back into the matrix directly, bypassing the ATP-synthase
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
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
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
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
is ATP an exothermic or endothermic reaction?
endothermic; coupled with electron transport
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
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
what class of inhibitor is dinitrophenol?
a respiratory stimulant
what is the effect of dinitrophenol?
- increased metabolic rate
- high oxygen consumption
- high body temperature
which inhibitors act on complex I?
amytal and rotenone
which inhibitors act on cytochrome c reductase?
antimycin A
which inhibitors act on ATP synthase?
oligomycin
which inhibitors act on cytochrome c oxidase?
potassium cyanide
are you gona kill this exam tmro?
YES
who is going to get 100% in this test?
I AM
