mitochondrial function structure citric acid cycle electron transport regulatory/modulatory...
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Mitochondrial Function
• Structure
• Citric acid cycle
• Electron transport
• Regulatory/modulatory signaling
Mitochondrial Structure
• Principal metabolic engine
• Symbiotic bacteria– 6k-370kBP genome– Human: 13 proteins
• Dual membrane– ie: two bilayers– Outer membrane highly
permeable– Inner membrane highly
impermeable
Mitochondrial Matrix• Highly oxidative environment• Unique proton gradient
– High pH (8), negative (-180 mV), ~18 kJ/mole– H+ actively transported out of matrix
– H+ leak back as H+PO4 2-
• Capture gradient energy for ATP synthesis– H+ ATPase pump– ADP-ATP antiporter
• Other proton co-transporters– Pyruvate, citrate– Glutamate, citruline
Metabolic Substrates
• Sugars– Metabolized in cytoplasm to pyruvate– Co-transported to matrix with H+– Bound to Coenzyme A as Acetyl-CoA
• Fatty acids– To intermembrane space as Acyl-CoA– To matrix as Acyl-carnitine– Metabolized to Acetyl-CoA in matrix
• Proteins
CH3
C=O
COO-
Acetyl Coenzyme A
• Common substrate for oxidative metabolism
• S-linked acetate carrier
Oxygen
Coenzyme A
Carbon
Isocitrate
a-Ketoglutarate
Succinyl CoASuccinate
Fumarate
=
Malate
Oxaloacetate
CoA
CoA
CoA
NADH +
NADH+ GTP
FADH2
NADH
The Citric Acid Cycle
Citrate
Acetyl-Coenzyme A
These carbons will be removed
New carbons
Electron transport
• Couple NADH/FADH2 electrons to H+ export– Ideally this completes
– Electron leakage
NADH + H+ + ½ O2 NAD+ +H2ONAD+ + H++2e- NADH E0=-0.32V
½O2+2 H++ 2e- H2O E0=0.82V
KEGG pathway
KEGG http://www.genome.jp/kegg/pathway.html
Enzyme Commission (EC) number•Hierarchical•Function-centric nomenclature•Compare
•Gene Ontology (GO) ID•Entrez RefSeq•UniProt ID
Metabolite
Cyclic redox reactions
Oxidized
Reduced
NADH
FADH2
NAD+
FAD CoQ/ubiquinone
dihydroubiquinone
Cyto-C3+
Cyto-C2+
O2
H2O
NAD+ NADH E0 = -0.32VFAD FADH2 E0 = -0.22VUbuquinone E0 = 0.10VCytochrome C E0 = 0.22VO2 H2O E0 = 0.82V
NADH/Complex I
• Nicotinamide adenine dinucleotide– H dissociates as H-
• Complex I (NADH reductase)• Then e- to ubiquinone
– 46 subunit protein– Nuclear-derived proteins– mtDNA-derived proteins– Transfer e- to ubiquinone– Shuttle 2 H+/e-
FADH2/Complex II
• Flavin Adenine Dinucleotide– H disrupts C-ring– Electron transfer flavoprotein
• Complex II– Succinate reductase
• Transfers 2H• from succinate to FAD
• No H+ transport
– ETF-ubiquinone oxidoreductase
• Transfers 2H• from FADH2 to ubiquinone
Complex III and IV
• Ubiquinone (UQ)– 2 electron carrier
• Complex III (cytochrome reductase)– Transfer e- from ubiquinone to cytochrome c– Coupled with H+ transport– Rieske “2Fe2S” redox center– 1 Ub to 2 CyC
http://bcs.whfreeman.com/stryer/pages/bcs-main.asp?s=00010&n=99000&i=99010.01&v=category&o=&ns=0&uid=0&rau=0
Complex IV
• Cytochrome oxidase– Transfer 1x e- from cytochrome C to oxygen– Coupled with H+ transport– 4x cytochrome yield 2xH2O
• Transport complex– Supercomplex of I, III, IV– Stoichiometry of 1:2:4– Transport 8 e- and 36 H+ per citrate– ATP?
Mitochondrial membrane potential
• Few ion channels
• Low H+– High H+ flux/H+ current– Proton equilibrium potential ~+50 mV– ~ -100 mV relative to cytoplasm
• H+ coupled transport– Malate, pyruvate, glutamate, Ca, Pi
• Charge coupled transport– ATP:ADP exchange, Ca
Proton ATPase/Complex V
• ATP driven proton pump– “Reversible”– Couples H+ gradient to ATP synthesis
Mitochondrial control
• Mitochodrial nucleotide flux– Steady state (at rest), not equilibrium– Dynamic control
• Membrane potential
• Substrate+O2+ADPCO2+H2O+ATP
Substrate(Glucose,pyruvate,NADH)
O2
ADP
CO2
H2O
ATP
State 2 State 4Rest
State 5
Control by mitochondrial potential
• NADH oxidation coupled to H+ transport
• Greater , greater resistance, slower ox
Mitochondrial Depolarization
NA
DH
co
nte
nt
Mitochondrial uncouplingpoison
Leyssens et al., 1997
Extreme mitochondria redox states
• Substrate+O2+ADPCO2+H2O+ATP
• State 1: substrate & ADP limited
• State 2: substrate limited
• State 3: enzyme limited– Maximal activity
• State 4: ADP & Pi limited– Rest
• State 5: O2 limited
– <5-10 uM O2 ~ 15-30 mmHg, 2-4%
Control by mitochondrial redox state
• Cytochrome oxidase (complex IV)– Iron (heme) redox centers (Fe2+/Fe3+)– Copper redox center (Cu+/Cu2+)
• Redox centers more oxidized– More O2
– Less ADP
Hoshi, et al., 1993
State 3
State 4Cu
Fe
E=E0 - RT/nF ln(Qprod/Qreac)
Redox cascade backs up by accumulation of product
Extreme mitochondria redox states
• State 1: substrate & ADP limited– Electron transport chain oxidized– High
• State 2: substrate limited– ETC oxidized, low
• State 3: enzyme limited– ETC reduced, low
• State 4: ADP & Pi limited– ETC reduced, high
• State 5: O2 limited– ETC reduced, high
Control by calcium
• Calcium activated enzymes– Pyruvate dehydrogenase– Oxoglutarate dehydrogenase– NAD+-isocitrate dehydrogenase – F0F1
• Membrane potential– Na-Ca antiporter– Ca uniporter depolarization with
cytoplasmic Ca• Reduce F0F1 efficiency• Increase NADH oxidation
Electron leakage
• Ubiquinone rapidly releases e-– Radical formation: O2
•
– Bypasses electron transport• Complex I• Complex III
• Oxidative damage– Thiol crosslinking, DNA damage, etc– Inhibition of Complex I & III– Buffered by intermembrane GSH, Mn-SOD