Respirasomes

Functional combination of two or more electron-transfer complexes

Respirasome of complex III & IV

Cardiolipin (abundant in inner mito membrane) critical to formation of respirasomes

Conservation of the e- Transfer Energy in a Proton Gradient

Standard free energy change of e- from NADH to O2

NADH + H+ + ½ O2 NAD+ + H2O G’o = -nFE’o = -220 kJ/mol

Oxidation of succinate G’o = -150 kJ/mol

Using the energy to pump protons out of the matrix 4H+ (complex I), 4H+ (complex III), 2H+ (complex IV)

; NADH + 11 HN+ + 1/2O2 NAD+ + 10Hp

+ + H2O

Conservation of the e- Transfer Energy in a Proton Gradient

Electrochemcial energy proton-motive force Energy stored in proton gradient Two components

Chemical potential energy : separation of chemical H+ Electrical potential energy : separation of charge

Proton-Motive Force

Free energy change for the creation of electrochemical gradient by ion pump

G = RTln(C2 / C1) + ZF C : concentrations of an ion, C2 > C1

Z : absolute value of electrical charge (1 for a proton) transmembrane difference in electrical potential

ln(C2 / C1) = 2.3 (log [H+]P - log [H+]N)

= 2.3 (pHN – pHP) = 2.3 pH

G = 2.3 RTpH +F Active mitochondria

; 0.15 ~ 0.2 V, pH = 0.74 G = 19 kJ/mol (of H+)

~ 200 kJ/mol (NADH)

Reactive oxygen species (ROS)

ROS generation of oxidative phosphorylation

- Radical •Q- intermediate generated during complex I QH2

QH2 complex III

Pass an electron to O2

O2 + e- •O2- (superoxide)

- Detoxification systems Superoxide dismutase (SOD) Glutathione peroxidase (GPx)

Oxidation of NADH in plant mitochondria

Analogous to mito ATP synthesis mechanism of animal Plant specific alternative mechanism

Regeneration of NAD+ from unneeded NADH

Direct e- transfer from ubiquinone to O2 (bypassing complex III and complex IV)

Energy from e- transfer heat generation

CN- alternative QH2 oxidase

Oxidative Phosphorylation19.2 ATP Synthesis

Chemiosmotic Model

“ The electrochemical energy inherent in the difference in proton concentration and the separation of charge across the inner mitochondrial membrane – the proton-motive force – drives the synthesis of ATP as protons flow passively back into the matrix through a proton pore associated with ATP synthase”

Chemical potential∆ pH

Electrical potential∆ ψ

Coupling of Electron Transfer & ATP Synthesis

Experiment to demonstrate ‘coupling’

ADP + Pi

succinate(1) Substrate oxidation(2) O2 consumption(3) ATP synthesis

Isolated mitochondria

Experiment 1

(1) (ADP + Pi) addition no respiration & ATP synthesis

(2) Succinate addition respiration & ATP synthesis

(3) CN- addition inhibiting respiration & ATP synthesis

Coupling of electron transfer & ATP synthesis

Coupling of Electron Transfer & ATP Synthesis

Experiment 2

(1) Succinate addition no respiration & ATP synthesis(2) (ADP + Pi) addition respiration & ATP synthesis(3) Oligomycin or venturicidin addition inhibiting respiration & ATP synthesis(4) DNP addition continuing respiration without ATP synthesis (uncoupling)

Further demonstration of coupling of electron transfer & ATP synthesis

Chemical uncouplers 2,4-dinitrophenol (DNP) &

carbonylcyanide-ρ-trifluoromethoxyphenylhydrazone (FCCP) Weak acids with hydrophobic properties

Release protons in the matrix dissipation of proton gradient

Ionophores Valinomycin (peptide ionophore binding K+) Transport of inorganic ions through

membranes dissipation of electrical gradient

Coupling of Electron Transfer & ATP Synthesis

Evidence for the Role of a Proton Gradient in ATP Synthesis

Artificial generation of electrochemical gradient

Leads to ATP synthesis without oxidizable substrate

Mechanism of ATP formation

1. Karl Lohman (1929)2. Fritz Lipmann (1953)3. Efraim Racker (1960)4. Peter Mitchell (1961)5. Masasuke Yoshida (1997) 6. Paul D. Boyer

How is the pmf transmitted to the ATP synthesis?

FoF1-ATPase

ATP Synthase

Mitochondrial ATP synthase (complex V) F-type ATP synthase Similar to ATP synthase of chloroplast

and bacteria Two functional domains F1 : peripheral membrane protein

ATP synthesis Isolated F1 : ATP hydrolysis

(originally called F1 ATPase) Fo (o : oligomycin-sensitive)

Membrane integrated Proton pore

ATP Synthase : F1

33(9 subunits) Knoblike structure with alternating and arrangement subunits; catalytic sites for ATP synthesis subunit

Central shaft Association with one of the three subunits (-empty)

Induction of conformational difference in subunits difference in ADP/ATP binding sites of subunits-ATP, -ADP, -empty

ATP Synthase : Fo

ab2c10-12 (3 subunits) C subunit

Small (Mr 8,000), hydrophobic two transmembrane helices Two concentric circles

Inner circle : N-terminal helices Outer circle (55 Å in diameter) : C-terminal helices

Mechanism of ATP synthesis in F1

18O-exchange experiment with purified F1

Incubation of purified F1 with ATP in 18O-labelled water

Analysis of 18O incorporation into Pi 3 or 4 isotopes in Pi

Repetitive reaction of both ATP hydrolysis and ATP synthesis

; ADP + Pi ATP + H2O, G’o ≈ 0 reversible reaction!

Mechanism of ATP synthesis in F1

Kinetic study for confirmation of G’o ≈ 0 Enz-ATP Enz-(ADP + Pi)

K’eq = k-1/k1 = 24 s-1/10 s-1 = 2.4 G’o ≈ 0 differ from ATP (free in solution) hydrolysis

G’o = -30.5 kJ/mol (K’eq=105) FoF1 has high affinity to ATP (Kd < 10-12 M) than ADP (Kd ≈ 10-5 M) 40 kJ/mol

difference in binding energy Equilibrium toward ATP synthesis

Release of ATP from the enzyme surface ; major energy barrier (not ATP formation) Proton gradient makes it possible