The Chemiosmotic Theory Explains The Mechanism Of Oxidative Phosphorylation

Mitchell's chemiosmotic theory postulates that the energy from oxidation of components in the respiratory chain is coupled to the translocation of hydrogen ions (protons, H+) from the inside to the outside of the inner mitochondrial membrane. The electrochemical potential difference resulting from the asymmetric dis-

Malonate

Succinate -

Complex I

NADH-

FMN, FeS

Uncouplers

Oligomycin

Complex II

FAD FeS

Complex I

FMN, FeS

Uncouplers

Oligomycin

Piericidin A Amobarbital Rotenone

Carboxin TTFA

BAL Antimycin A

H2S CO CN-

Complex III

Piericidin A Amobarbital Rotenone

BAL Antimycin A

Complex III

Cyt b, FeS, Cyt c1 -*—

I

Uncouplers

Uncouplers

Complex IV |

H2S CO CN-

Complex IV |

Cyt a

Cyt a3

Cu

Oligomycin

Figure 12-7. Proposed sites of inhibition (Q) of the respiratory chain by specific drugs, chemicals, and antibiotics. The sites that appear to support phosphorylation are indicated. BAL, dimercaprol. TTFA, an Fe-chelating agent. Complex I, NADH:ubiquinone oxidoreductase; complex II, succinate:ubiquinone oxidoreductase; complex III, ubiquinol:ferricytochrome c oxidoreductase; complex IV, ferrocytochrome c:oxygen oxidoreductase. Other abbreviations as in Figure 12-4.

tribution of the hydrogen ions is used to drive the mechanism responsible for the formation of ATP (Figure 12-8).

The Respiratory Chain Is a Proton Pump

Each of the respiratory chain complexes I, III, and IV (Figures 12-7 and 12-8) acts as a proton pump. The inner membrane is impermeable to ions in general but particularly to protons, which accumulate outside the membrane, creating an electrochemical potential difference across the membrane (A|H+).This consists of a chemical potential (difference in pH) and an electrical potential.

A Membrane-Located ATP Synthase Functions as a Rotary Motor to Form ATP

The electrochemical potential difference is used to drive a membrane-located ATP synthase which in the presence of P; + ADP forms ATP (Figure 12-8). Scattered over the surface of the inner membrane are the phos-phorylating complexes, ATP synthase, responsible for the production of ATP (Figure 12-1). These consist of several protein subunits, collectively known as F1, which project into the matrix and which contain the phosphorylation mechanism (Figure 12-8). These sub-

units are attached to a membrane protein complex known as F0, which also consists of several protein sub-units. F0 spans the membrane and forms the proton channel. The flow of protons through F0 causes it to rotate, driving the production of ATP in the F1 complex (Figure 12-9). Estimates suggest that for each NADH oxidized, complex I translocates four protons and complexes III and IV translocate 6 between them. As four protons are taken into the mitochondrion for each ATP exported, the P:O ratio would not necessarily be a complete integer, ie, 3, but possibly 2.5. However, for simplicity, a value of 3 for the oxidation of NADH + H+ and 2 for the oxidation of FADH2 will continue to be used throughout this text.

Experimental Findings Support the Chemiosmotic Theory

(1) Addition of protons (acid) to the external medium of intact mitochondria leads to the generation of ATP.

(2) Oxidative phosphorylation does not occur in soluble systems where there is no possibility of a vectorial ATP synthase. A closed membrane must be present in order to achieve oxidative phosphorylation (Figure 12-8).

(3) The respiratory chain contains components organized in a sided manner (transverse asymmetry) as required by the chemiosmotic theory.

Atp Synthesis With Chemiosmotic Theory

OUTSIDE

Figure 12-8. Principles of the chemiosmotic theory of oxidative phosphorylation. The main proton circuit is created by the coupling of oxidation in the respiratory chain to proton translocation from the inside to the outside of the membrane, driven by the respiratory chain complexes I, III, and IV, each of which acts as a proton pump. Q, ubiquinone; C, cytochrome c; F1, F0, protein subunits which utilize energy from the proton gradient to promote phosphorylation. Uncoupling agents such as dinitrophenol allow leakage of H+ across the membrane, thus collapsing the electrochemical proton gradient. Oligomycin specifically blocks conduction of H+ through F0.

OUTSIDE

Figure 12-8. Principles of the chemiosmotic theory of oxidative phosphorylation. The main proton circuit is created by the coupling of oxidation in the respiratory chain to proton translocation from the inside to the outside of the membrane, driven by the respiratory chain complexes I, III, and IV, each of which acts as a proton pump. Q, ubiquinone; C, cytochrome c; F1, F0, protein subunits which utilize energy from the proton gradient to promote phosphorylation. Uncoupling agents such as dinitrophenol allow leakage of H+ across the membrane, thus collapsing the electrochemical proton gradient. Oligomycin specifically blocks conduction of H+ through F0.

Table 12-1. States of respiratory control.

State 1 State 2 State 3

State 4 State 5

Conditions Limiting the Rate of Respiration

Availability of ADP and substrate Availability of substrate only The capacity of the respiratory chain itself, when all substrates and components are present in saturating amounts Availability of ADP only Availability of oxygen only

The Chemiosmotic Theory Can Account for Respiratory Control and the Action of Uncouplers

The electrochemical potential difference across the membrane, once established as a result of proton translocation, inhibits further transport of reducing equivalents through the respiratory chain unless discharged by back-translocation of protons across the membrane through the vectorial ATP synthase. This in turn depends on availability of ADP and P;.

Uncouplers (eg, dinitrophenol) are amphipathic (Chapter 14) and increase the permeability of the lipoid inner mitochondrial membrane to protons (Figure 12-8), thus reducing the electrochemical potential and short-circuiting the ATP synthase. In this way, oxidation can proceed without phosphorylation.

Which Way Does Atp Synthase Spins
ATP

If!

J!

Outside \

V

C

C

'C

/

Mitochondrial inner membrane

Mitochondrial inner membrane

Figure 12-9. Mechanism of ATP production by ATP synthase. The enzyme complex consists of an F0 sub-complex which is a disk of "C" protein subunits. Attached is a Y-subunit in the form of a "bent axle." Protons passing through the disk of "C" units cause it and the attached Y-subunit to rotate. The Y-subunit fits inside the F, subcomplex of three a- and three p-sub-units, which are fixed to the membrane and do not rotate. ADP and P| are taken up sequentially by the p-subunits to form ATP, which is expelled as the rotating Y-subunit squeezes each p-subunit in turn. Thus, three ATP molecules are generated per revolution. For clarity, not all the subunits that have been identified are shown—eg, the "axle" also contains an e-subunit.

THE RELATIVE IMPERMEABILITY OF THE INNER MITOCHONDRIAL MEMBRANE NECESSITATES EXCHANGE TRANSPORTERS

Exchange diffusion systems are present in the membrane for exchange of anions against OH- ions and cations against H+ ions. Such systems are necessary for uptake and output of ionized metabolites while preserv ing electrical and osmotic equilibrium. The inner bilipoid mitochondrial membrane is freely permeable to uncharged small molecules, such as oxygen, water, CO2, and NH3, and to monocarboxylic acids, such as 3-hydroxybutyric, acetoacetic, and acetic. Long-chain fatty acids are transported into mitochondria via the carnitine system (Figure 22-1), and there is also a special carrier for pyruvate involving a symport that utilizes the H+ gradient from outside to inside the mitochondrion (Figure 12-10). However, dicarboxylate and tri-

OUTSIDE N-Ethylmaleimide

Inner mitochondrial membrane

INSIDE

H2PO4-N-Ethylmaleimide Hydroxycinnamate

Pyruvate-

Malate2

a-Ketoglutarate2

ADP3

Atractyloside

HPO4

Malate2

Malate2

ATP4

Figure 12-10. Transporter systems in the inner mitochondrial membrane. ©, phosphate transporter; D, pyruvate symport; (D, dicarboxylate transporter; D, tricarboxylate transporter; DD, a-ketoglutarate transporter; DD, adenine nucleotide transporter. W-Ethyl-maleimide, hydroxycinnamate, and atractyloside inhibit (D ) the indicated systems. Also present (but not shown) are transporter systems for glutamate/aspar-tate (Figure 12-13), glutamine, ornithine, neutral amino acids, and carnitine (Figure 22-1).

carboxylate anions and amino acids require specific transporter or carrier systems to facilitate their passage across the membrane. Monocarboxylic acids penetrate more readily in their undissociated and more lipid-solu-ble form.

The transport of di- and tricarboxylate anions is closely linked to that of inorganic phosphate, which penetrates readily as the H2PO^ ion in exchange for OH-. The net uptake of malate by the dicarboxylate transporter requires inorganic phosphate for exchange in the opposite direction. The net uptake of citrate, isocitrate, or «'s-aconitate by the tricarboxylate transporter requires malate in exchange. a-Ketoglutarate transport also requires an exchange with malate. The adenine nucleotide transporter allows the exchange of ATP and ADP but not AMP. It is vital in allowing ATP exit from mitochondria to the sites of extramito-chondrial utilization and in allowing the return of ADP for ATP production within the mitochondrion (Figure 12-11). Na+ can be exchanged for H+, driven by the proton gradient. It is believed that active uptake of Ca2+ by mitochondria occurs with a net charge transfer of 1 (Ca+ uniport), possibly through a Ca2+/H+ antiport. Calcium release from mitochondria is facilitated by exchange with Na+.

Inner

OUTSIDE mitochondrial INSIDE membrane

Inner

OUTSIDE mitochondrial INSIDE membrane

Ca2 Exchange Antiport Neuron

Figure 12-11. Combination of phosphate transporter ((J)) with the adenine nucleotide transporter ((J) in ATP synthesis. The H+/P, symport shown is equivalent to the P/OH- antiport shown in Figure 12-10. Four protons are taken into the mitochondrion for each ATP exported. However, one less proton would be taken in when ATP is used inside the mitochondrion.

Figure 12-11. Combination of phosphate transporter ((J)) with the adenine nucleotide transporter ((J) in ATP synthesis. The H+/P, symport shown is equivalent to the P/OH- antiport shown in Figure 12-10. Four protons are taken into the mitochondrion for each ATP exported. However, one less proton would be taken in when ATP is used inside the mitochondrion.

Ionophores Permit Specific Cations to Penetrate Membranes

Ionophores are lipophilic molecules that complex specific cations and facilitate their transport through biologic membranes, eg, valinomycin (K+). The classic uncouplers such as dinitrophenol are, in fact, proton ionophores.

A Proton-Translocating Transhydrogenase Is a Source of Intramitochondrial NADPH

Energy-linked transhydrogenase, a protein in the inner mitochondrial membrane, couples the passage of protons down the electrochemical gradient from outside to inside the mitochondrion with the transfer of H from intramitochondrial NADH to NADPH for intramitochondrial enzymes such as glutamate dehydrogenase and hydroxylases involved in steroid synthesis.

Oxidation of Extramitochondrial NADH Is Mediated by Substrate Shuttles

NADH cannot penetrate the mitochondrial membrane, but it is produced continuously in the cytosol by 3-phosphoglyceraldehyde dehydrogenase, an enzyme in the glycolysis sequence (Figure 17-2). However, under aerobic conditions, extramitochondrial NADH does not accumulate and is presumed to be oxidized by the respiratory chain in mitochondria. The transfer of reducing equivalents through the mitochondrial membrane requires substrate pairs, linked by suitable dehydrogen-ases on each side of the mitochondrial membrane. The mechanism of transfer using the glycerophosphate shuttle is shown in Figure 12-12). Since the mitochon-drial enzyme is linked to the respiratory chain via a flavoprotein rather than NAD, only 2 mol rather than 3 mol of ATP are formed per atom of oxygen consumed. Although this shuttle is present in some tissues (eg, brain, white muscle), in others (eg, heart muscle) it is deficient. It is therefore believed that the malate shuttle system (Figure 12-13) is of more universal utility. The complexity of this system is due to the impermeability of the mitochondrial membrane to oxalo-acetate, which must react with glutamate and transami-nate to aspartate and a-ketoglutarate before transport through the mitochondrial membrane and reconstitution to oxaloacetate in the cytosol.

Ion Transport in Mitochondria Is Energy-Linked

Mitochondria maintain or accumulate cations such as K+, Na+, Ca2+, and Mg2+, and P;. It is assumed that a primary proton pump drives cation exchange.

NAD+

CYTOSOL

Glycerol 3-phosphate

Dihydroxyacetone phosphate

OUTER MEMBRANE

Glycerol 3-phosphate

Dihydroxyacetone phosphate

INNER MEMBRANE

MITOCHONDRION

Glycerol 3-phosphate -

fad

GLYCEROL-3-PHOSPHATE DEHYDROGENASE (MITOCHONDRIAL)

I

- Dihydroxyacetone "

Respiratory chain

Figure 12-12. Glycerophosphate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion.

The Creatine Phosphate Shuttle Facilitates Transport of High-Energy Phosphate From Mitochondria

This shuttle (Figure 12-14) augments the functions of creatine phosphate as an energy buffer by acting as a dynamic system for transfer of high-energy phosphate from mitochondria in active tissues such as heart and skeletal muscle. An isoenzyme of creatine kinase (CK^) is found in the mitochondrial intermembrane space, catalyzing the transfer of high-energy phosphate to cre-atine from ATP emerging from the adenine nucleotide transporter. In turn, the creatine phosphate is trans ported into the cytosol via protein pores in the outer mitochondrial membrane, becoming available for generation of extramitochondrial ATP.

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Responses

  • berardo
    How the chemiosmotic theory explains p/o ratios?
    5 years ago
  • Mathilda
    How is the electrical potential across the membrane generated for chemiosmosis?
    5 years ago
  • virginio
    What are the mechanism of atp production by chemiosmosis?
    5 years ago
  • MICHELLE
    What are the theories of Oxidative phosphorylation?
    3 years ago
  • Judy
    What is chemiosmotic phosphorylation?
    11 months ago

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