ENERGY COUPLING or ENERGY TRANSDUCTION

 

The free energy released by electron transport (ET) must be conserved in a form that ATP synthase can utilize.

 

Chemiosmotic Theory

 

·        1961, Peter Mitchell

 

·        ET ® coupled to the pumping of H+ from mitochondrial matrix across the inner membrane to outside ® create an H+ electrochemical potential gradient across the inner membrane ® this gradient is the energy source which drives the ATP synthesis.

 

·        Fig. 17-20.

 

 

SUPPORTING EVIDENCE FOR CHEMIOSMOTIC HYPOTHESIS

 

·        Require intact inner mitochondrial membrane.

 

·        Inner membrane impermeable to diffusion by ions such as H+, OH-, K+ and Cl-. 

 

·        ET results in the transport of H+ from matrix to outside of inner membrane, creating a considerable electrochemical gradient across the inner membrane.

 

·        The measured membrane potential is 0.168 V (more negative inside).

 

·        The pH of matrix is 0.75 unit higher than that of the intermembrane space.

 

·        The DG’ for the transport of a H+ out of matrix is 21.5 kJ mol-1.

 

·        Compounds that increase the permeability of the inner membrane to H+ allow ET to proceed but inhibit ATP synthesis.  Hence, oxidation (or ET) and phosphorylation can be uncoupled by UNCOUPLERS such as 2,4-dinitrophenol (DNP).

P/O Ratios:  relate the amount of ATP synthesized to the amount of oxygen reduced.

 

P/2e Ratios:  relate the amount of ATP synthesized per 2-e reduction of some electron acceptor other than oxygen.

 

E Donor

E Acceptor

Agent(s) added

P/O  or

P/2e

Succinate (or FADH2)

1/2   O2

 

2

Succinate (or FADH2)

1/2   O2

Amytal

2

Succinate (or FADH2)

1/2   O2

CN-

0

Succinate (or FADH2)

1/2   O2

DNP

0

Succinate (or FADH2)

Fe(CN6)3-

 

1

Succinate (or FADH2)

Fe(CN6)3-

CN-

1

b-Hydroxybutyrate (or NADH)

1/2   O2

CN-

0

b-Hydroxybutyrate (or NADH)

Fe(CN6)3-

CN-

2

 

 

In addition:

 

·        ATP is synthesized by mitochondria or chloroplasts when a pH gradient is artificially generated without ET.

 

·        Bacteriorhodopsin is a purple membrane protein from halobacteria.  It pumps H+ when illuminated.  Synthetic lipid vesicles containing the bacteriorhodopsin and beef heart mitochondrial ATPase can synthesize ATP when illuminated.  (Walther Stoeckenius and Efraim Racker)

 

·        Using intact mitochondria and submitochondrial particles (inner membrane inside out), the NADH-Q reductase, Q-Cyt c reductase, Cyt c, Cyt c oxidase, and ATP synthase have all been shown to be asymmetrically oriented. 

 

 

MECHANISM OF ATP SYNTHESIS

 

·        See Figs. 17-21b and 17-25 for E. coli proton-pumping  ATP synthase (= F1F0 ATPase) structure.

 

·        Discover Fo and F1 by Efraim Racker.

 

·        F1 is a water-soluble peripheral membrane protein consisting of (a3b3gde). Active in ATP synthesis in intact ATP synthase assembly but only active in ATP hydrolysis in isolated soluble form.

 

·        F0 is a water-insoluble transmembrane protein consisting of a1b2c9-12 in E. coli and some additional subunits in mitochondrial F0.  Forms the proton channel.

 

·        Binding Change Mechanism by Paul Boyer.

 

·        See Fig. 17-26 for mechanism of ATP synthesis.  Note: (1) Conformational changes take place when the T site is occupied by ATP and the L site is occupied by ADP and Pi.  (2) Such conformational changes result in the conversions of L to T, T to O, and O to L.  This conformational change requires energy, which is transmitted to the catalytic a3b3 assembly via the rotating ge assembly shown in green in Fig. 17-26.  (3) ATP is synthesized at the new T site and the original bound ATP is released from the new O site.