Aerobic respiration

Aerobic respiration• Mitochondrial structure and function

– Visible under light microscope

– Universal in aerobic eukaryotes

– Have own DNA and ribosomes

– Number and shape vary widely in different cell types

• Number: more in cells with higher E requirements

• Shape: can undergo fission and fusion to yield typical ‘cylinder’ shape or more complex tubular networks


– Membranes

• Outer: permeable to many things

– Porins, large central pore

• Inner: highly impermeable

– Rich in cardiolipin (also present in bacterial membanes)

– Channels for pyruvate, ATP, etc


– Membranes

• Outer: permeable to many things

– Porins, large central pore

• Inner: highly impermeable

– Rich in cardiolipin (also present in bacterial membanes)

– Channels for pyruvate, ATP, etc

• Cristae

– Complex invaginations of the inner membrane

– Functionally distinct

– Joined to inner membrane via narrow channels


– Intermembrane space• Between inner and outer membranes• Also within the cristae• Acidified ( high [H+] ) by action of the Electron Transport Chain (ETC)

– H+ are pumped from matrix into this compartment– ATP synthase lets them back into the matrix


– Matrix• Compartment within the inner membrane• Very high protein concentration ~500mg/ml• Contains:

– ribosomes and DNA– Enzymes of TCA cycle, enzymes for fatty acid degradation

• Glycolysis– 6C glucose --> --> --> 2x3C pyruvate + 2NADH

• Glycolysis– 6C glucose --> 3C pyruvate + 2NADH

NADH enters the mitochondriaby one of two mechanisms:

1. aspartate-malate shuttle NADH --> NADH2. glycerol phosphate shuttle

NADH --> FADH2

• Pyruvate to TCA

The Aspartate – Malate Shuttle

• TCA cycle– 3C --> 2C + CO2 + NADH– CoA derived from pantothenic acid

– Condensation: 2C + 4C --> 6C– Isomerization


– Condensation: 2C + 4C --> 6C– Isomerization

– 6C --> 5C + CO2 +NADH

– 5C --> 4C + CO2 +NADH

– 4C + GTP

– 4C + FADH2


– Condensation: 2C + 4C (OA) --> 6C– Isomerization

– 6C --> 5C + CO2 +NADH

– 5C --> 4C + CO2 +NADH

– 4C + GTP

– 4C + FADH2– Hydration

– 4C (OA) + NADH

• TCA cycle 2Pyruvate + 8NAD+ + 2FAD + 2GDP + 2Pi --> 6CO2 + 8NADH + 2FADH2 + 2GTP

– Adding in products of glycolysis, 2NADH + 2ATP

– Total yield of glycolysis and TCA cycle: 8NADH + 4FADH2 + 4ATP

Fatty acid catabolism• Enzymes localized to mitochondrial matrix

– Fatty acids cross inner membrane and become linked to HS-CoA– Each turn of cycle generates FADH2 + NADH2 + Acetyl-CoA

Amino acid catabolism• Enzymes in matrix

– AA’s cross inner membrane via specific transporters– Enter TCA at various points

General outline of oxidative phosphorylation

Oxidation-reduction potentials• Reducing agents give up electron share

– The lower the affinity for electrons, the stronger the reducing agent• NADH is strong, H2O is weak

• Oxidizing agents receive electron share– The higher the affinity for electrons, the stronger the oxidizing agent

• O2 is strong, NAD+ is weak

• Couples– NAD+ - NADH couple (weak oxidizer, strong reducer)– O2 - H2O couple (strong oxidizer, weak reducer)

Oxidation-reduction potentials• Eo’ measures affinity for electrons

– Negative Eo’ indicates a stronger reducing agent

– Positive Eo’ indicates a stronger oxidizing agent

– In Electron Transport Chain: e- are passed from stronger reducing agents to form weaker reducing agents

• Pass from more negative to more positive Eo’

• H2O is the weakest reducing agent (of interest here)

• NADH --> H2OG0’ = -53kcal/mol

7ATP(max), ~3ATP(real)

Electron Transport Chain• Electron carriers

– Flavoproteins• FAD/FMN (riboflavin)

– NADH DH (complex I)– Succinate DH (complex II, TCA cycle)




– Cytochromes• Heme (Fe) groups




– Cytochromes• Heme (Fe) groups

– Cu atoms• Cu2+ <--> Cu1+

– Ubiquinone• Free radical intermediate• Lipid soluble• Dissolved within inner mitochondrial membrane

– Fe-S centers




– Cytochromes (b, c1, c, a)• Heme (Fe) groups

– Cu atoms• Cu2+ <--> Cu1+

– Ubiquinone (Q or UQ)• Free radical intermediate• Lipid soluble• Dissolved within inner mitochondrial membrane

– Fe-S centers

Electron Transport Chain• Complex I passes e- from NADH to UQ and pumps 4H+ out of matrix• Complex II passes e- from FADH2 to UQ• UQ shuttles e- to Complex III

Electron Transport Chain• Complex III passes e- to Cytochrome c and pumps 4H+ out of matrix• Cytochrome c passes e- to Complex IV• Complex IV passes e- to O2 forming H2O and pumps 2H+ out

1 pH unit diff

ATP synthesis: The ATP Synthase enzyme• F1 head/sphere (ATPase) catalyzes ADP + Pi <--> ATP• F0 base embedded in inner membrane (H+ pass through this)• F0 + F1 = ATP synthase

– The two pieces are connected via two additional proteins• Central rod-like gamma subunit• Peripheral complex that holds F1 in a fixed position

– Location• Bacteria = plasma mem• Mitochondria = inner mem• Chloroplast = thylakoid

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Intermembrane space

matrix

H+

ATP

The ATP Synthase mechanism• Binding Change Mechanism

– E of H+ movement is used to force release of ATP from enzyme• Enz-ADP + Enz-Pi --> Enz-ATP G0’ ~ 0

– Each F1 active site progresses through three distinct conformations• Open (O), Loose (L), Tight (T)• Conformations differ in affinity for substrates and products

– Central gamma () subunit rotates causing conformation changes

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Rotational catalysis by ATP synthase

• If true, should be able to run it backwards (ATP --> ADP + Pi) and watch gamma spin like a propeller blade

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Other fxns of electrochemical gradient

• E also used for:– Import of ADP + Pi (+H+) and export of ATP– Import of pyruvate (+H+)

• Uncoupling sugar oxidation from ATP synthesis– Uncoupling proteins (UCP1-5)

• UCP1/thermogenin, shuttles H+ back to matrix (endothermy)– Brown adipose tissue

» Present in newborns (lost with age) and hibernating animals» Generates heat

– 2,4-dinitrophenol (DNP)• Ionophore that can dissolve in inner membrane and shuttle H+ across

– 1930’s stanford diet pill trials: overdose causes a fatal fever

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Aerobic respiration