Mitochondrial function and dysfunction Flashcards
functions of mitochondria
- oxidation of fat, protein and CHO for energy
- steroid hormones and neurotransmitter synthesis
- nucleotide synthesis
- calcium buffering
- growth and proliferation
- apoptosis and cell growth
anatomy of mitochondrion
- outer membrane
- intermembrane space
- innermembrane
- matrix
- cristae junction
- F0, F1 complexes
- DNA
- ribosomes
what is the mitochondria inner membrane permeable to
O2, CO2 and H2O
what is the mitochondria outer membrane permeable to
<5000 Da
3 stages of cellular respiration
- production of acetyl CoA (link reaction)
- oxidation of acetyl Coa
= TCA cycle - electron transport and chemiosmosis
how is acetyl CoA produced
from pyruvate in the link reaction
TCA Cycle
- Acetyl CoA (from link)
- citric acid
- isocitric acid
- alpha-ketoglutaric acid
- succinyl CoA
- Succinate
- Fumarate
- Malate
- oxaloacetate
- acetyl CoA
where in TCA is NADH produced
- isocitric acid -> alpha-ketoglutaric acid
- alpha-ketoglutaric acid -> succinyl CoA
- malate -> oxaloacetate
= 3 x NADH
Where in TCA is FADH2 produced
- succinate -> fumarate
= 1 x FADH2
Where is CO2 produced in the TCA cycle
- isocitric acid -> alpha-ketoglutaric acid
- alpha-ketoglutaric acid -> succinyl CoA
= 2 x CO2
where is ATP produced in the TCA cycle
- succinyl CoA -> succinate
= 1 x ATP
reduced NAD
NADH
Steps of ETC
- Complex I takes 2e- from NADH. Energy released is used to pump 4H+ across the membrane
- complex II takes 2e- from FADH2. No H+ is pumpd across the membrane
- Ubiquinone (Q) takes 2e- from complex I and II and transfers to complex III
- Complex III accepts these 2e- from Q. Energy released is used to pump H+ across the membrane
- Cyt C takes e- from complex III and transfers to complex IV
- Complex IV accepts e- from Cyt C
- cycle repeats and Complex IV accumulates 4e-
- 4e- used to reduce molecular oxygen to water
what transfers electrons from Complex I and II to complex III
ubiquinone (Q)
what does ubiquinone do in ETC
transfers e- from Complex I and II to complex III
Where does complex I take it 2e- from
NADH
Where does complex Ii take it 2e- from
FADH2
Where does complex III get its 2e- from
Ubiquinione, Q
what does Cyt C do
takes e- from complex III to complex IV
where does complex IV get its e- from
Cyt c, from complex IV
What happens when complex IV accumulates 4e-
4e- used to reduce molecular oxygen to water
which complexes pump H across membrane
I, III and IV
where to pumped protons go
enter ATPase to produce ATP via chemiosmosis
where is Q
inner mitochondrial membrane
where is Cyt C
intermembrane space
where is Complex I
inner mitochondrial membrane
where is complex II
mitochondrial matrix
where is complex III
inner mitochondrial membrane
where is complex IV
inner mitochondrial membrane
where is ATPase
inner mitochondrial membrane through to matrix
list electron carriers
- NAD+
- flavoprotens
- iron-sulphur clusters
- Ubiquinone
- cytochromes
NAD+ as electron carrier
- accepts 2e- and one H+ = NADH
flavoproteins as electron carriers
accept 1e- in semiquinone form or 2e-, and 2 H+
- FAD -> FADH2
- FMN -> FMNH2
iron sulphur clusters as electron carriers
- Fe-S accept and release one e- at a time
non haem prosthetic group associated with flavin enzymes.
Fe2+ or Fe3+ ; net charge is somewhere inbetween as electrons are dispersed amount Fe
uniquinone as electron carrier
aka coenzyme Q10
- only electron carrier not bound to protein complex
- freely diffusable in the non-polar interior of the IMM
cytochromes as electron carrier
c1, c, a and a3
- capable of absorbing visible light due to haem group
- haem prosthetic group oscialte between Fe2+ and Fe3+ after acceptin an electron
- cytochromes carry one e-
- cyt c is mobile
what is complex I
NADH dehydrogenase
role of complex I
- oxidises NADH from TCA cycle, glycolysis and FA oxidation
- reduces Q for rest of ETC
- transports H+ across IMM to support ATP synthesis
- major contributor to cellular reactive oxygen species productive and oxidative stress
structure of complex I
NADH dehydrogenase integral membrane enzyme composed of: - 9 redox cofactors - 44 different subunits has a membrane arm and a matrix arm - many iron-sulphur centres - FMN containing flavoprotein
how does complex I transfer electrons from NADH to Q
- FMN in the matrix arm accepts 2e- from NADH converting it to reduced form FMNH2
- Fe-S clusters in the matrix arm transfer 2e- protein N2 in membrane arm, one at a time
- electrons transfer from N2 through membrane arm to Q
- Q is reduced to QH2
- 2H+ are pulled from the matrix
function of complex I in ETC
catalyses two simultaneous and coupled processes
- takes 2e- from NADH and passes them to Q, pulling 2H+ from matrix to generate QH2
- transfers 4H+ from matrix to intermembrane
what is complex III
cytochrome c oxidoreductase
where does QH2 go
complex III
Structure of Complex III
- cytochrome c oxidoreductase
- membrane protein
- dimer of identical monomers
- each monomer has 11 different subunits
- Cyt C1 and Rieske of Complex III project into the IMS and interact with CytC
- has 2 distinct binding sites of QH2
examples of some of the different subunits found in each monomer of complex III
- cytochrome b, with 2 haem groups
- rieske, Fe2S2 protein
- cytochrome C1, with heam group
what are the two binding sites on complex III for QH2
QN and QP
summary equation of the Q cycle
QH2 + 2Cytc (Fe3+) + 2H+ (m) -> Q + 4H+ (ims) + 2Cytc (Fe2+)
QH2 + 2H+ -> Q + 2e- + 4H+
what is the Q cycle
Oxidation of QH2 by Cytochrome C, catalysed by complex III. Protons are also pumped across the IMM
Structure of Cytochrome C
- small
- single haem group and single e-
- 12kDa
- 104 amino acids
what does leakage of cytochrome C out of mitochondrial memebrane trigger
apoptosis
what is complex IV
cytochrome C oxidase
structure of complex IV
cytochome C oxidase
- 14 protein subunits
- 2 catalytic subunits
- 2 haems: Cyt a and Cyt a3
- 2 copper centres: CuA and CuB
what is sepcial about complex IV
it reduces oxygen without generating superoxide // free radicals
process of complex IV actions
- two molecules of reduced Cyt C each donate an e- to CuA
- first e- passes through haem group of subnits 1 to the Fe-CuB centre to reduce copper
- second e- stops at haem a3, reducing Fe3+ to Fe2+
- oxygen now recruited, forming haem a3-oxygen complex
- proximity of reduced Cu to the haem complex reduces it to copper peroxied which bridges haem and Cu
- Cyt C delivers 3rd e- which cleaves O-O peroxide bond, with help of 2 matirix H+ = generation of Fe4+ with haem a3
- Cyt c delivers final e- which reduces Fe4+ to Fe3+
- 2 more H+ release 2 molecules of water and reset the system
- Complex IV also pumps 4H+ into IMM
overall reaction at complex IV
4cyt c (reduced) + 8H+ + O2 -> 4Cyt c (oxidised) + 2H2O + 4H+
How many H+ does complex IV remove from the matrix
8
- removes 4 by chemical reaction to form water
- pumps 4 across membrane into IMM
What is compelx II
succinate dehydrogenase
how many protons does complex II pump
none
which complex pumps no protons
complex II
structure of complex II
succinate dehydrogenase, has 4 subunits - SDH-A : FAD as a proton acceptor - SDH-B: 3 Fe-S clusters - SDH-C: cytochrome b - SDH-D: cytochrome b also has Q binding site
role of Complex II
Involved in production of FADH2, via TCA cycle
takes 2e- from FADH2 to give to Q
what is the overall purpose of thee ETC
to generate proton gradient to drive ATP synthase
what powers the ETC
Redox reactions
- simultaneous reduction and oxidation resulting in transfer of electrons
what is a redox potential
the measure of ease with which a molecule will accepy protons
- more positive the redox potential, the more readily a molecule is reduced
why does ETC happen via lots of small reactions
lots of little reactions each produce a small amount of energy.
this is favourable becuase it is easier to control than one large sum of energy that would be produced if reaction went straight from NADH -> O2
PMF
proton motive force
what forces are involved in the electrochemical gradient of H+ across the IMM
- large force due to membrane potential
- smaller force due to [H+] gradient
what is complex V
F1F0 ATP synthase
structure of complex V
F1F0ATP synthase
- 500kDa complex which makes use of electrochemical gradient
- F0 is hydrophobic unit in membrane with 10 identical subunits
- F1 is hydrophillic catalytic unit with 3 identical alpha beta subunits
what drives complex V
spins at 150 Hz
- flow of H+ down electrochemical grad drives the F0 roto that lies in the membrane
- H+ bind to empty F0 subunits
- once protonated F0 subunits complete a full circle, protons exit the matrix
what is the energy conversion at complex V
energy stored in proton gradient is converted to rotational energy
rotatory catalysis model
- when F1 is in the open state, ADP and P enter the active site
- protein then closes around ADP and P and binds them loosely
- protein undergoes another conformational change forcing ADP and P closer together
= AS is now in a T-state to produce ATP with very high affinity - AS goes back to open which releases the ATP and binds more ADP and P for next cycle
what happens if F1 doesnt have ADP and P attached
F1 will not allow F0 and stalk to rotate
- very important for respiratory control
total ATP made from oxidative phosphorylation
26-28
- 3-5 from glycolysis (via NADH)
- 5 from link reaction (via NADH)
- 18 from TCA cycle (via NADH and FADH2)
types of mitochondrial dysfunction
- impairment of ETC and ATPsynthesis machinery
- inadequate no. of mitochondria
- accumulation of damaged mitochondria
what causes impairment of ETC and ATPsynthesis machinery
single enzme disorder or ROS/RNS
RNS and ROS
reactive nitrogen species
reactive oxygen species
what causes inadequate no. of mitochondria
imparied mitochondrial dynamics / biogenesis
what causes accumulation of damaged mitochondria
impaired mitophagy
what is the most common single enzyme mitochondrial disorder
complex I disorder
complex I disorder
- most common single enzyme mitochondrial disorder
- mutations discovered in 26/44 genes, 7mtDNA and 21nDNA
- range of symptons and severity
- 50% fatal under 2yo
- 75% fatal under 10yo
what does complex I disorder causes
- lactic acidosis which is fatal bc of sever inhibiton of ETC
- mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS)
- Leigh syndrome
MELAS
mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes
what is heteroplasmy
presence of >1 type of mitochondrial genome
- multiple copies of mtDNA per cell and not segregated like nuclear genome
- proprtion of mutant DNA vary between indivuduals and tissues
- bottleneck at myosis - few mitochondria transmitted so variable mutaton burden in offspring
how does diabetes affect mitochondria metabolism
- causes marked inhibition of mitochondrial metabolism in pancreatic beta cells
- mitochondrially-generated ATP stimulates insulin secretion and senses glucose
- mouse model of T2D showed reduced mitochondrial metabolism and ATP function
what are the major source of ROS
mitochondria
what are the most likely origins of ROS in intact cells
complex I and III
how are ROS produced
leak of e- as they progress from donor redox centres to molecular oxygen (ETC)
- premature single e- reduction of molecualr oxygen earier in the ETC forms superoxide radical
ROS production
highlight reactive •OH generated from O2•- and H2O
- initiates formation of lipid radicals and lipid peroxyl radiocals
- mitochondria are also exposed to RNS (NO•) which can react with O2•- to form peroxynitrate ONOO-
Superoxide anion
O2•-
what do O2•- and NO• form
peroxynitrate ONOO-
how many types of DNA to mitochondria have
2
- nuclear DNA
- mitochondrial DNA
mitochondrial DNA
- maternally inherited
- multiple copies per mitochondrion
- located in The matrix
- encodes critical subunits of the ETC
what do ROS do to mitochondrial DNA
cause base pari lesions which accumulate with age
- H2O2 forms reactive •OH which causes formation of 8-oxoG from purines
- produces G-T inversions on replication
Mitochondrial maintenace
methods of minimising dysfunction
- fusion and fission
- ROS scavenging
- mitophagy
ROS scavenging
removal of ROS
O2•- is converted to H2O2 by superoxide dismutase
mitochondrial dynamics
fission and fusion
fission: required for cell division, increased itochondrial number and segregation of damaged mitochondrial
fusion: allows mitochondria contents to mix and may inrease ATP production
Fission
required for cell division, increased itochondrial number and segregation of damaged mitochondrial
Fusion
allows mitochondria contents to mix and may inrease ATP production
steps of fission
- cystolic Drp1 is phosphorylated
- Drp1 recruited to mitochondria and binds receptors
- forms cuff around mitochondria which constricts the organelle
- constriction severs both membranes
- new mitochondria generated
steps of fusion
- Mfn1 and 2 localise o the outer membranes and dock the two mitochondrion together by fusion of outer membrane
- OPA 1 localised on inner membrane, responsible for fusing the two inner membranes together
- shares content of mitochondria and dilutes the effects of any damage
what is OPA 1 used for
localised on he inner membrane of mitochondria to fuse the two inner membranes together during fusion
where else is OPA 1 critical
ETC function and apoptosis
- OPA1 bridges the entrance to the cristae within the mitochondrion
- disruption of OPA1 bridge disrupts the ETC and releases CytC
- Cyc C escapes the mitochondrion and promotes apoptosis
quality control of mitochondria
mitophagy
mitophagy
quality control of mitochondria
- selective mitochondrial degradation/recycling
- usually upregulated in response to stress
healthy mitochondria
PINK1 degraded by proteomsomes
Depolarised mitochondria
PINK1 and parkin act on the outer MM proteins to recruit them into autophagosomes for recycling
pathologies of mitochondiral dysfunction
Complex II : huntington’s
Complex IV: Alzheimer’s
PINK1/Parkin: parkinson;s
ROS: autism
chronic fatigue, diabetes, epilipsy, cerebral palsy, muscular dystrophy, cardiomyopathy and more
where is cytochrome c reoxidised
Complex IV of the Electron Transport Chain
Which components of the Electron Transport Chain are the most likely producers of Reactive Oxygen Species (ROS) in mitochondria
Complexes I and III
