Oxidative phosphorylation 19.1-19.3

Oxidative phosphorylation

e transfer reaction in mitochondrion

-outer mm-pores→ free diffusion of small molecules

inner mm -tight→ no free passage

e transfer between e carriers

Mitochondrial matrix

Enclosed by an inner mm → contains pyruvate dehydrogenase complex + enzymes of the citric acid cycle, fatty ox pathway and amino acid ox → ONLY glycolysis which occur in the cytosol

Respiratory chain

Electrons enter a series of electron carriers - e are transported by NADH and FADH2

3 types of e transfer occur in ox phosp

1. Driect transfer of e as in Fe3+→ Fe2+

2. Transfer as a hydrogen atom (H+ +e-)

3. Transfer as a hydride ion (:H-) carrying 2 e-

Electron carrying molecules

• NAD

• FLavoproteins

• Quionone - coenzyme Q

• Cyrochromes

• Fe-S proteins

Ubiquinone Q

• Lipid soluble benzoquionone with a long side chain

• Can accept one e to become semiquinone radical

• Or 2 electrons to becom ubiquinol

• Freely diffusible in the lipid bilayer

Cyrochromes

=proteins with characteristic strong absorption of visible light- heme groups

three classes: a, b and c → different light absorption spectra

Fe-S proteins

Iron (not in heme) with sulfur atoms or with sulfur of Cys residues in the protein or both

Participate in one electron transfer- one iron atom of the cluster is oxidized or reduced

Resipratory chain electron carriers

E move from NADH, succinate, or other primary electron donor _> flavoproteins, W, Fe-S proteins and cytochromes to O2

Is e transfer favorable?

Yes! Its spontaneous:

NADH → Q → cytochrome b →

cytochrome c1 → cytochrome c →cytochrome a → cytochrome a3 → O2

E flows from carriers of low E’ to higher E’

COmplex 1= NADH dehydrogenase

1. Catalyze transfer of hydride ion to Q from NADH (exergonic)

2. 4 H+ is pumped to intermm space (endergonic) (against a proton gradient)

   NADH+5H+ (N)+Q→NAD+ + QH2 + 2H+ (P)

Prosthetic groups= FMN containing flavoprotein, Fe-S

L shape

Proton pump is driven by e transfer

Complex 2=succinat dehydrogenase

Catalyze electron transfer from succinate (ox) to Q (reduced)

Prosthetic groups= FAD, Fe-S

NO proton pump

(Functions in the citric acid cycle)

2 transmembrane subunits C and D

COmplex 3= Ubiquinone: cyt c oxidoreductase/cyt bc1

Carries e from reduced Q to Cyt c

Prosthetic groups: Heme, Fe-S

Proton pump

Dimer- functional unit

Cyt c

Small soluble problem

Heme group accepts e from complex 3→ moves to the intermembrane space to complex 4 to donate e to a binuclear Cu center

Complex 4= Cytochrome oxidase

Transfers e from cyt c to O2→ H2O

2 Cu ions with SH groups of 2 Cys residues

Pump protons

Heme groups

Cyt C - > CuA→ heme a→ heme a3→ CuB→ O2

Overall reaction: 4 cyt (red) + 8H+N + O2 → 4 cyt c (ox) + 4H+P + 2H2O

Q cycle

Q cycle is a process occurs in complex 3→ helps transfer e from QH2 (2xe) to cyt c (only carry 1 e)

QH2→ carries 2 e→ Q cycle splits the e to different paths→ efficient transfer

Never a release of the radical formed!

Stage 1: E moves from QH2 (ox) → complex 3 + release H+ at one side

Stage 2: Other side→ Q is reduced and protons are taken up

O2→ H2O

Final electron acceptor

4 e + 4 H+→ heme a3 and Cu can only take one e at a time→ O2 is reduced stepwise to water → intermediate forms like O2- or H2O2

Risk of escaping and forming ROS

Respirasome

Copmplex 1,3,4

Complex 2- free floating in the membrane

3 enzymes that reduce Q to QH2 in the inner mitochondrial membrane

• Acyl-CoA dehydrogenase via ETF (electron transferring flavoprotein) and ETF:Q oxidoreductase (from β-oxidation),

• Glycerol 3-phosphate dehydrogenase (from glycerol metabolism or glycolysis),

• And dihydroorotate dehydrogenase (from pyrimidine synthesis).

Proton motive force

=PMF

=concentration gradient (due to H+) + electrical gradient ( separation of charge)

Equation 2 e- through the respiratory chain

2 NADH + 2H+ + O2 → NAD+ + 2H2O

Highly exergonic

ROS

Superoxide

Hydrogen peroxide

Hydroxyl radicals

Energy coupling

PMF drives the synthesis of ATP as protons flow passively back into

the matrix through a proton pore in ATP synthase

Coupling= obligate connection between ATP synthesis and e flow through respiratory chain - neither can proceed without the other

Coupling process

ADP + Pi + succinate →

1. substrate is oxidized to fumarate

2. O2 is consumed

3. ATP is synthesized

   Ozygen consumption and ATP synthesis depend on the presence of the substrate! + ADP + Pi

Block ATP synthase

Inhibitors blocking e transfer→ block ATP synthase

Ex oligomycin- block proton channel of ATP synthase→ e transfer stops

Uncoupling through phosphorylation

Uncouple oxidation from phosphorylation

Still catalyze e transfer from succinate to NADH + O2 but no ATP synthesis

Example compounds: DNP, FCCP

ATP synthase

F type ATPase

ADP + Pi → ATP driven by flow of protons from P→ N

2 components: F0 (into membrane, proton pore) and F1 (peripheral membrane protein, make ATP)

How does the enzyme compensate for the unfavorable energy of ADP-Pi→ ATP

The binding energy of enzyme ATP complex compensate for the unfavorable energy of the reaction

ATP synthase stabilizes ATP relative to ADP + Pi by binding ATP more tightly releasing enough energy to counterbalance the cost of making ATP

Binds ATP with very high affinity and ADP with low affinity→ this drives the reaction to produce ATP

DIfference between typical enzyme and ATP synthase

Typical E: catalyze by lowering activation energy

ATP synthase: Uses a proton gradient to rotate parts of its structure→ mechanical energy converted to chemical energy y synthesizing ATP. Major energy barrier is the release of ATP from E not the reaction

Transport of ADP

) Transport of ADP -> matrix
- ATP/ADP antiport
*) driven by PMF
*) Transport of Pi
- symport with H+

Inner mitochondria mm

Adenine nucleotide translocase binds ADP3—> transports into matrix and exchange for ATP4- transported out

The proton motive force drives ATP-ADP exchange

Phophate translocase- promotes symport of one H2PO4 and one H+

Transport of NADH formed in cytosol to matrix in mito

• Through the malate-ASP shuttle

• “Loss less”

• NADH reduces OAA→ Malate

• Malate crosses inner mitochondrial membrane via transporter

• Malate is oxidized back to OAA→ NADH in the matrix

• NADH to ETC

• OAA cant cross the membrane

• The shuttle doesnt transport NADH directly but it electrons

Glycerol-3-phosphate shuttle

• Transport in skeletal muscle and brain

• ) Transport of e from NADH formed i cytosol
  ) e- are directly transferred to QH2
  -> fewer protons are pumped across the inner mm
  
  Bypass complex 1!! in this pathway
  Not loss less!
  
  Contribution to PMF is smaller! only 6 protons
  instead of 10!

Summary oxidative phosphorylation

SUMMARY:
*) 30-32 ATP per glucose -> 976 kJ/mol -> 34% efficiency! Is it good enough? The rest of energy goes of as heat!

*) Thermogenin (UCP1)

*) Thermogenin (UCP1)
- "brown fat"- contain a high density of mitochondria
Contain a lot of heme-> color
Infants has more brown fat-> to keep warm

Regulation of oxidative phosphorylation

- [ADP] is high -> electron transport is very active HIGH
- If [ATP] is high-> e transport goes DOWN