The Proton-Motive Force CHAPTER 21 The Proton-Motive Force

Figure 21.1: Chemiosmotic hypothesis: electron transport via the respiratory chain pump protons into intermembrane space. These protons can be used to do work in making ATP as they are transported back into the matrix through ATPase. It takes 3 H+ transported into the matrix to make one ATP molecule

Complex V: ATP Synthase F0F1 ATP Synthase uses the proton gradient energy for the synthesis of ATP Structure is composed of a “knob-and-stalk” structure F1 (knob) contains the catalytic subunits for ATP production F0 (stalk) has a proton channel which spans the membrane Passage of protons through the F0 (stalk) into the mitochondrial matrix is coupled to ATP formation Estimated passage of 3 protons (H+) per ATP synthesized

Figure 21.3 Knob-and-stalk structure of ATP synthase A molecular machine Intermembrane space H+ enter from the Intermembrane space into subunit a. Binding of this H+ induces a clockwise rotation of the c ring and the g subunit. rotation of the g subunit also induces conformational changes in the a3b3 hexamer A second H+ waiting in the lower channel of subunit a is released into the matrix. Figure 14.14 The knob-and-stalk structure of ATP synthase. (a) The F1 component is on the inner face of the membrane. The F0 component, which spans the membrane, forms a proton channel at the a–c interface. The passage of protons through this channel causes the rotor (shaded) to rotate relative to the stator. The torque of these rotations is transmitted to F1 where it is used to drive ATP synthesis. (b) Molecular structure of the rotor and a portion of the stator of ATP synthase of Saccharomyces cerevisiae. The study of many molecular structures like this, based on X-ray crystallography, assisted in deduction of the complete model shown in (a). [PDB 1QO1]. Matrix

Figure 21.3 Knob-and-stalk structure of ATP synthase A molecular machine Intermembrane space The binding site for the ADP/ATP lies in the clefts between adjacent a and b subunits in the a3b3 cylinder. The active site for ATP synthesis is mostly part of the b subunits One 120o rotation of the c-g subunits requires the translocation of one proton. Figure 14.14 The knob-and-stalk structure of ATP synthase. (a) The F1 component is on the inner face of the membrane. The F0 component, which spans the membrane, forms a proton channel at the a–c interface. The passage of protons through this channel causes the rotor (shaded) to rotate relative to the stator. The torque of these rotations is transmitted to F1 where it is used to drive ATP synthesis. (b) Molecular structure of the rotor and a portion of the stator of ATP synthase of Saccharomyces cerevisiae. The study of many molecular structures like this, based on X-ray crystallography, assisted in deduction of the complete model shown in (a). [PDB 1QO1]. Matrix

Figure 21.7: A closer look at subunits c and a. H+ enters from intermembrane space Figure 21.9 The pH is lower on the intermem. side vs. matrix Protonation of the Asp residue makes it more hydrophobic. This induces a change in the c unit were the Asp unit Interacts more with the membrane. As this change occurs a rotation of one subunit c occurs. When a protonated Asp moves into the Matrix half channel it is released due to the lower pH the result is that the negative Asp now interacts more with the subunit a half channel H+ exit into the matrix space

Figure 21.8: Proton movement across the membrane

Figure 21.8: Proton movement across the membrane Negative charged Asp

Figure 21.8: Proton movement across the membrane Neutral Asp Each proton that passes results in a 120 degree rotation of c subunit.

The binding Change Mechanism The mechanism of ATP synthesis from ADP and Pi The binding Change Mechanism Open: new ATP is released and ADP and Pi bind 2. Loose: bound ADP and Pi cannot be released 3. Tight: condensation of ADP and Pi is favored to form ATP. The ATP formed is very tightly bound. Figure 14.15 Binding change mechanism of ATP synthase. The different conformations of the three catalytic sites are indicated by different shapes. ADP and Pi bind to the yellow site in the open conformation. As the shaft rotates in the counter-clockwise direction (viewed from the cytoplasmic/matrix end of the F1 component), the yellow site is converted to a loose conformation where ADP and Pi are more firmly bound. Following the next step of the rotation, the yellow site is converted to a tight conformation and ATP is synthesized. Meanwhile, the site that had bound ATP tightly has become an open site, and a loose site containing other molecules of ADP and Pi has become a tight site. ATP is released from the open site, and ATP is synthesized in the tight site. Figure 21.4

Figure 21.5 The binding Change Mechanism The mechanism of ATP synthesis from ADP and Pi Figure 21.5 The binding Change Mechanism Follow the yellow region of one b subunit.

Figure 21.16 Transport of ATP, ADP and Pi across the Inner mitochondrial membrane - Once ATP is made, it must be sent out to the cytosol and more ADP and Pi must be transported into the matrix. Adenine nucleotide translocase: unidirectional exchange of ATP for ADP (antiport) Start here Figure 14.17 Transport of ATP, ADP, and Pi across the inner mitochondrial membrane. The adenine nucleotide translocase carries out unidirectional exchange of ATP for ADP (antiport). The symport of Pi and H+ is electroneutral.

Figure 21.16 Transport of ATP, ADP and Pi across the Inner mitochondrial membrane - Antiport of Pi and OH- occurs resulting in an electrical neutral translocation. (or symport with a H+) Figure 14.17 Transport of ATP, ADP, and Pi across the inner mitochondrial membrane. The adenine nucleotide translocase carries out unidirectional exchange of ATP for ADP (antiport). The symport of Pi and H+ is electroneutral.

Figure 21.16: The Energy change regulates the use of fuels 1 H+ translocated 3 H+ per ATP synth. 10 H+ per O reduced

Regulation of Oxidative phosphorylation Overall rate of oxidative phosphorylation depends upon substrate availability and cellular energy demand Important substrates: NADH, FADH2, ADP Resting state: ADP levels low NADH and FADH2 are not oxidized via elec. transport CAC slows down Oxidative Phosph. Slows down Active state: ADP levels rise NADH and FADH2 begin being oxidized via elec. transport CAC is more active Oxidative Phosph. increases Electrons do not flow to O2 unless ATP is in demand

P:O Ratio = molecules of ADP phosphorylated atoms of oxygen reduced The relationship between oxygen consumption (respiration) and ATP synthesis (phosphorylation) The P:O Ratio P:O Ratio = molecules of ADP phosphorylated atoms of oxygen reduced Translocation of 3 H+ are required by ATP synthase for each ATP produced 1 H+ is needed for transport of Pi, ADP and ATP NET: 4 H+ transported for each ATP synthesized and transported

Calculation of the P:O ratio Complex I III IV #H+ translocated/2e- 4 4 2 Recall that two species supplied 2 e- each for proton translocation: NADH and succinate (FADH2) For NADH: 10 H+ translocated/O (2 e-) P/O = (10 H+/4 H+) = 2.5 ATP/O For succinate: (FADH2  QH2) substrate = 6 H+/O (2 e-) P/O = (6 H+/4 H+) = 1.5 ATP/O

Glycerol 3-phosphate shuttle One method electrons from cytoplasmic NADH produce ATP Figure 21.11 The glycerol 3-phosphate shuttle. Electrons from NADH can enter the mitochondrial electron-transport chain by reducing dihydroxyacetone phosphate to glycerol 3-phosphate. Electron transfer to an FAD prosthetic group in a membrane-bound glycerol 3-phosphate dehydrogenase reoxidizes glycerol 3-phosphate. Subsequent electron transfer to Q to form QH2 allows these electrons to enter the electron-transport chain.

Malate – Aspartate shuttle Another method electrons from cytoplasmic NADH produce ATP Figure 21.12 The malate-aspartate shuttle.

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