Mitochondrial Function Structure Citric acid cycle Electron transport Regulatory/modulatory signaling.

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