what does oxidative phosphorylation do
converts energy derived from oxidation of glucose, fatty acids, and amino acids to ATP's high-energy phosphate bonds
where does oxidative metabolism take place
function and location of the electron transport chain?
oxidizes energy, from oxidation of fuels in TCA cycle and B-oxidation of fatty acids, conserved in NADH and FADH2,
via a series of enzymes embedded in mito inner membrane
what is final acceptor of H's from NADH and FADH2 in the etc?
how does structure of the mito impact ETC?
outer membrane is permeable to everything
folded inner membrane contains respiratory/ETC and its enzymes - which are linked to enzymes that make ATP, the ATP synthesis enzymes
how are electrons transferred, broadly, in mito resp chain?
e- are transferred from NADH/FADH2 via ubiquinone, coenzyme Q, and cytochromes to molecular O2
molecular O2 is reduced to H2O
energy from oxidation of NADh and FADH2 by O2 is used to produce ATP from ADP + Pi by ATP synthase, FoF1 ATPase complex
what is chemiosmotic model
explains how energy from transport of electrons to O2 is transformed into the high energy bonds of ATP
what kind of rxns cause transfer of e- in the etc?
oxidation-reduction steps as electrons pass through complexes of proteins that span the mito inner membrane
what generates electrochemical gradient across mito inner membrane?
as e- pass through complexes in inner mito membrane, pumping of protons from mito matrix to cytosolic side of inner mito membrane
what comprises delta-P?
electrochemical gradient across mito inner membrane
= a membrane potential beacuse of pumping positive charges out of the mito
AND a proton gradient b/c of pumping protons out of the mito
what is the ratio of H+ pumped/e- passing through for each of the complexes of the etc?
Complex I: 4H+ pumped per electron
Complex III: 2H+ per electron
Complex IV: 4H+ per electron
this generates electrochemical gradient across membrane
structure of the ATP synthase?
a proton pore through the inner membrane and a catalytic head-piece that protrudes into the matrix
how does ATP synthase function?
protons that were pumped out during e- transport to O2 re-enter via the proton pore of the ATP synthase complex
this causes conformation change in catalytic headpiece
this change releases ATP bound to 1 site, and catalyzes formation of a new ATP from ADP + Pi at another site
the newly formed ATP is transferred to the bound site, will be released by next proton entering the proton pore
how can we categorize the process that the ATP synthase complex facilitates?
oxidative phosphorylation - transfer of e- and pumping of protons is coupled to synthesis of ATP
what defines the rate of respiration in the etc?
1) transfer of electrons
2) reduction of O2
what regulates the rate of respiration in the etc?
1) rate of ATP synthesis
3) other bioenergetic work functions which utilize the delta-P
what is difference in effect on etc action between if delta-P is utilized/not utilized?
if delta-P is not utilized, rate of e- transfer or O2 uptake decreases b/c of back pressure exerted on respiratory chain by high accumulated delta-p
if delta-p is utilized for ATP synthesis or bioenergetic work functions, the rate of e- transfer becomes faster in order to keep generating the delta-P
how does the permeability of the mito inner membrane impact the respiration rate?
membrane's impermeability to protons means that protons are pumped out only at discrete sites in complexes I, III, IV, and pumped back in through proton pore of ATP syntase (links oxidation-phosphorylation)
what can make mito inner membrane permeable?
what's the impact?
damage or chemical modification by uncoupling agents to mito inner membrane makes it leaky to protons
a delta-P will be immediately dissipated, since pumped-out protons non-specifically re-enter the mito rather than re-enter through proton pore of ATP synthase
what are uncoupling agents
proteins that form channels through the mito inner membrane for protons to pass from the intermembrane (cytosolic) space to the matrix
thus short-circuits the ATP synthase
what happens to the delta-P and respiratory chain in the presence of an uncoupling agent?
lose delta-p beacuse discharge the gradient by carrying protons back into the mitochondria
respiratory chain still is working, so get movement of e- through the chain, but do not make any ATP
this uncouples phosphorylation and oxidation
associated w/ heat production in brown adipose tissue
important for infants to maintain body temp and for animals to hibernate
in skeletal muscle
drug target for obesity - idea that if find activators of UCP3 to "waste" the delta-P as heat rather than use it to support fat synthesis reactions
what is only part of the etc that isn't protein-bound?
how is ubiquinone synthesized?
from an intermediate produced during cholesterol synthesis
what is complex I? what does it contain? what does it do?
Complex I: NADH Dehydrogenase or NADH:Coenzyme Q oxidoreductase complex
contains: iron-sulfur (FeS) proteins, FMN (flavin mononucleotide)
e- pass from NADH -> FMN -> FeS centers -> ubiquinone (Q)
this reduces QH2, reduced ubiquinone
Complex II name?
What does it contain?
What does it do?
Complex II = Sucicnate Dehydrogenase (TCA cycle enzyme)
e- from succinate pass to FAD to Q, produces QH2
what processes do e- from FADH2 come from?
how do they enter the etc?
FADH2 are produced by B-oxidation of fatty acids and by a-GP shuttle
e- from FADH2 pass on to Q via electron transfer flavoprotein
are protons pumped out of mito by complex II?
how many e- do NADH and FADH2 transfer to Q?
rate? result of this?
Q can accept e- 1 at a time
this produces the ubisemiquinone radical before the HQ radical accepts the 2nd electron to produce QH2, reduced ubiquinone
what are the cytochromes?
proteins containing hemes in which the iron in the heme unergoes oxidation-reduction
what is the name of Complex III?
what does it contain?
what is its action?
Complex III: B:c1 complex
Contains: cytochromes b and c1 and iron-sulfur centers
QH2 passes 1 e- at a time to Cyt b (Fe 3+) to produce reduced Cyt b (Fe2+) which reduces the FeS, which reduces Cyt C1, which reduces Cyt c (Fe3+) to reduced Cyt C (Fe2+
b:c1 complex pumps 2 protons out/electron
what is complex IV?
what does it contain?
what is its action?
Complex IV: Cytochrome Oxidase complex
Contains: Cytochromes a and a3 and 2 major copper centers, CuA and CuB
Reduced Cyt C passes 1 e- at a time through Complex IV: reduces CuA, then Cyt A, then CuB, then Cyt a3
how many e- are needed to reduce molecular O2 to H2O?
when does this occur?
O2 + 4H+ + 4e- -> H2O
this occurs via complex IV, means that complex IV has 4 H+ pumped out/e- passing through
how does the ATP synthase complex work?
protons enter the proton pore in the top from the cytosolic side of the mito inner membrane
conformational changes occur in several subunits, causing rotations in the F1 rotor component
rotations discharge bound ATP and catalyze phosphorylation of ADP
this produces a new ATP by the alpha subunit
how much energy does oxidation of 1 mole of NADH and FADH2 produce?
NADH: 53 kCal/mol = 7-8 ATPs theoretically, 2.5 ATP actually
FADH2: 41 kCal/mol = 6 ATPs theoretically, 1.5 ATP actually
thus about 25-30% of available energy from oxidation of NADh and FADH2 is trapped as ATP
is the respiratory chain reversible? why/why not?
cannot synthesize O2 from H2O
because delta-G is strongly negative
what can delta-P power be used for besides ATP synthesis?
1. energized calcium uptake into the mito
2. adenine nucleotide translocase, exchanges ADP for ATP
3. reversed electron transport
4. transhydrogenase activity
energy-dependent reactions, which mito can carry out using proton motor force!
what is transhydrogenase activity
energy-dependent reduction of NADP+ to NAD+ and NADPH
in presence of a delta-P, equilibrium of this changes from 1, favors reduction of NADP+ by NADH, results in NADPH utilization for biosynthetic rxns
why's it important that delta-P can be used for Ca2+ uptake into the mito?
Ca2+ is toxic, so Ca2+ is removed from teh cytosol, stored in the ER and the mito
this requires a delta-P to pump Ca2+ into the mito
what is reversed e- transfer?
FADH2 reducing NAD+ to produce NADH rather than NADH reducing FAD/FMN
what is the effect of bioenergetic reactions that use delta-P, but are not the respiratory chain?
1. they compete w/ ATP for the delta-P
2. they speed up the rate of e- transfer by utilizing the delta-P
what inhibits complex I?
many chemicals; often rotenone is used
in rotenone presence, reduced FMN and iron sulfur centers of Complex I accumulate
e- thus are not passed to Q, and to O2
delta-P is not maintained -> ATP is not synthesized
what inhibits complex III?
QH2 accumulates, and Cyt C isn't reduced
what inhibits complex IV?
cyanide (CN) and carbon monoxide (CO)
CN: reduced Cyt C accumulates
what inhibits the ATP synthase complex?
oligomycin and DCCD bind to complex, block the proton pore
thus external protons can't enter the ATP synthase complex ->
ATP isn't synthesized ->
continuous proton pumping stops b/c of back pressure of accumulated cytosolic protons ->
O2 uptake slows down
what is respiratory control?
what controls its rate?
when is it slower, faster?
control of oxygen uptake by the delta-P aka rate of O2 uptake in presence of a respiratory chain substrate plus ADP + Pi relative to rate in presence of substrate only
O2 uptake in substrate only is low since delta-P generated isn't being used
O2 uptake in presence of substrate plus ADP + Pi is rapid b/c delta-P is being used to produce ATP
what is the cause of mitochondrial diseases re: resp chain?
cytochrome b, 3 of 6 protein subunits of cyt. oxidase, 7 of subunits of Cojplex I, and 2 subunits of the FoF1 complex come from mito proteins encoded by mito DNA
many mitochondrial diseases rsult from mutations either in the nuclear DNA or mito DNA encoding mito proteins
why is mito DNA prone to damage?
not protected by histones, as nuclear DNA is
are the major generator of reactive oxygen species
deficiencies in synthesis of parts of complexes assoc w/ mito DNA can be toxic
how is mito DNA inherited and trasmitted?
mito DNA is maternally inherited
transmitted by mother to children
examples of mito DNA lesions in disease?
parkinson's disease, cardiomyopathies, diabetes, several neurological disorders
what is respiratory control?
what determines it?
Respiratory control is control of the rate of O2 uptake in the respiratory chain by the proton motor force, and it is determined by the presence of substrate plus ADP + Pi relative to the rate of substrate only
O2 uptake is increased when you have ADP + Pi to turn into ATP, so that cells utilize O2 only if they would take it up anyways
What is effect on rate of oxygen uptake and rate of ATP synthesis in presence of Substrate, ADP, plus
2) an uncoupler
4) uncoupler + oligomycin
5) uncoupler + cyanide
Cyanide: no oxygen consumption because complex IV is blocked
An uncoupler: rapid oxygen consumption because proton motor force is discharged
Oligomycin: blocks proton core of complex V, ATP synthase, no ATP made but proton motor force functions
Uncoupler + oligomycin: same rate of O2 movement as in only uncoupler conditions; get no ATP production
Uncoupler + Cyanide: rate of O2 movement goes to 0 because cyanide poisons Complex IV
If Mito, substrate, ADP, Pi, and oxygen are available,
1) what would generate a high proton motive force?
2) what could be added to produce ATP?
- High proton motive force would be generated if add in malate + NADH
- Could add Ca2+ to produce ATP
why does rotenone inhibit the MA shuttle but not the a-GP shuttle?
Rotenone inhibits the MA shuttle but not the a-GP shuttle because the malate-aspartate shuttle uses NAD+, and rotenone inhibits complex I which oxidizes NADH to NAD+, whereas complex II uses FAD from succinate, and rotenone does not inhibit complex II, and the a-GP shuttle uses FAD+ to accept e- for transfer to Q
aka FADH2 enters beyond complex I
what woudl accumulate in the ETC in the presence of
1) antimycin A
- QH2 would accumulate in the presence of antimycin A since antimycin A inhibits Complex III
- Cytochrome C1 would accumulate in the presence of cyanide since CN inhibits Cytochrome C
what are mitochondrial work functions outside of the ETC that use the proton motive force?
energized Ca2+ uptake from the cyto to the matrix, transhydrogenase activity, adenine nucleotide translocase, reversed e- transport are mitochondrial work functions that can utilize the proton motive force
where do NADH and FADH2 used to set up the proton motive force come from?
NADH from TCA or glycolysis
FADH2 from TCA cycle