Lecture: 11‐11‐2016

CHAPTER 21 The Proton‐Motive Force Itapúa Binacional, on the border between Brazil and Paraguay, is one of biggest hydroelectric dams in the world.

Analogously, the mitochondrial The dam transforms enzyme ATP the energy of falling synthase transforms water into the energy of electrical energy. protons falling down an energy gradient into ATP. Chapter 21 Outline Definition: • The proton gradient generated by the

oxidation of NADH and FADH2 is called the proton‐motive force.

• Proton‐motive force (Δp) = chemical gradient (pH) + charge gradient (ΔΨ)

• The proton‐motive force powers the synthesis of ATP.

• Heterologous experimental systems confirmed that proton gradients can power ATP synthesis.

Peter Mitchell proposed the chemiosmotic hypothesis that was that electron transport and ATP synthesis are coupled by a proton gradient across the inner mitochondrial membrane. Chemiosmotic hypothesis

Electron transfer through the respiratory chain leads to the pumping of protons from the matrix to the cytoplasmic side of the inner mitochondrial membrane.

The pH gradient and membrane potential constitute a proton‐motive force that is used to drive ATP synthesis. Testing the chemiosmotic hypothesis

the respiratory chain An artificial system and ATP synthase are representing biochemically separate the cellular systems, linked only by respiration system a proton-motive force.

ATP is synthesized when reconstituted membrane vesicles containing bacteriorhodopsin (a light‐driven proton pump) and ATP synthase are illuminated.

The orientation of ATP synthase in this reconstituted membrane is the reverse of that in the mitochondrion F contains the proton • 0 ATP synthase is made‐up of two components. The F1 channel of the complex. component contains the active sites and overhangs into the mitochondrial matrix (Catalytic subunit)

• ATP synthases bind to one another to form dimers which then oligmerize. The oligomers contribute to the formation of cristae.

• Each enzyme has three active sites located on the three β subunits.

• The F0 component is embedded in the inner mitochondrial membrane and contains the proton ATP Synthase channel.

The F1 subunit consists of five types of • The γ subunit connects the F1 and F0 components. polypeptide chains (α3, β , γ, δ, and ε) • Each β subunit is distinct in that each subunit 3 interacts differently with the γ subunit. ATP synthase assists in the formation of cristae

(1) Electron transport generates a proton-motive force; (2) ATP synthesis by ATP synthase is powered by a proton-motive force.

The formation of oligomers of dimers of ATP synthase facilitates the formation of cristae, creating an area where the protons have ready access to the ATP synthase.

• The binding change mechanism accounts for the synthesis of ATP in response to proton flow.

• The three catalytic β subunits of the F1 component can exist in three conformations: Each of the β subunits is distinct because each interacts with a different face of the γ subunit.

• In the O (open) form, nucleotides can bind to or be released from the β subunit.

• In the L (loose) form, nucleotides are trapped in the β subunit.

• In the T (tight) form, ATP is synthesized from ADP and Pi. No two subunits are ever in the same conformation.

Each subunit cycles through the three conformations. The rotation of the γ subunit interconverts the β subunits.

Each 360-degree rotation of the γ subunit leads to the synthesis and release of 3 molecules of ATP. Direct observation of ATP‐driven rotation in ATP synthase

• It is possible to observe fluorescently labeled actin filament the rotation of the γ subunit directly.

• Cloned α3β3γ subunits were attached to a glass slide that allowed the movement of the γ subunit to be visualized as a result of ATP hydrolysis.

• The hydrolysis of a single ATP powered the rotation of the γ subunit Operates in a similar fashion in reverse gear 120o.

Answer Think of the discussion of enzyme-catalyzed reactions that the direction of a reaction is determined by the ΔG difference between substrate and products. An enzyme speeds up the rate of both the forward and the backward reactions. The hydrolysis of ATP is exergonic, and so ATP synthase will enhance the hydrolytic reaction. • Proton flow occurs through the F0 component of the ATP synthase.

• Subunit a, which touches the c ring, has two channels that reach halfway into the a subunit. One half channel opens to the intermembrane space and the other to the matrix.

• Protons enter the half channel facing the proton‐rich intermembrane space, bind to an glutamate residue on Proton one of the subunits of the c ring, and then leave the c channel subunit once they rotate around to face the matrix half channel.

• The force of the proton gradient powers rotation of the c ring.

• The rotation of the c ring powers the movement of the γ subunit, which in turn alters the conformation of the β subunits. Components of the proton‐conducting unit of ATP synthase

The c subunit consists of two helices that span the membrane. An glutamic acid residue in one of the helices lies on the center of the membrane. The structure of the a subunit appears to include two half‐channels that allow protons to enter and pass part way but not completely through the membrane Proton path through the membrane

The number of c rings determines the number of protons required to synthesize a molecule of ATP.

The c ring of vertebrates consist of 8 subunits, making vertebrate ATP synthase the most efficient known.

The movement of protons through the half‐channels from the high proton concentration of the inner membrane space to the low proton concentration of the matrix powers the rotation of the c ring. Proton motion across the membrane drives the rotation of the c ring

A proton enters from the intermembrane space into the cytoplasmic half‐channel to neutralize the charge on an aspartate residue in a c subunit. With this charge neutralized, the c ring can rotate clockwise by one c subunit, moving an aspartic acid residue out of the membrane into the matrix half‐channel. This proton can move into the matrix, resetting the system to its initial state. An overview of oxidative phosphorylation

Electron transport generates a proton-motive force that powers ATP synthase to synthesize

Answer: The number of c subunits is significant because it determines the number of protons that must be transported to generate a molecule of ATP. ATP synthase must rotate 360 degrees to synthesize three molecules of ATP; so, the more c subunits there are, the more protons are required to rotate the F1 units 360 degrees. 1) Glycerol 3‐phosphate shuttle

In muscle, electrons from cytoplasmic NADH can enter the electron‐ transport chain by using the glycerol phosphate shuttle.

When cytoplasmic NADH transported by the glycerol 3-phosphate shuttle is oxidized by the respiratory chain, 1.5 rather than 2.5 molecules of ATP are formed. 2) Malate‐aspartate shuttle

• In heart and liver, electrons from cytoplasmic NADH are used to generate mitochondrial NADH in malate‐aspartate shuttle.

• The malate‐aspartate shuttle consists of two membrane transporters and four enzymes. • The ATP‐ADP translocase enables the exchange of cytoplasmic ADP for mitochondrial ATP.

• ADP must enter the mitochondria for ATP to leave.

• The translocase is powered by the proton‐motive force, The mechanism of mitochondrial ATP‐ADP translocase

The translocase catalyzes the coupled entry of ADP into the matrix and the exit of ATP from it. The binding of ADP (1) from the cytoplasm favors eversion of the transporter (2) to release ADP into the matrix (3). Subsequent binding of ATP from the matrix to the everted form (4) favors eversion • The inner mitochondrial membrane has many transporters or carriers to enable the exchange of ions or charged molecules between the mitochondria and cytoplasm.

• The ATP‐ADP translocase, the phosphate carrier and the ATP synthase are associated with one another to form a large complex, the ATP synthasome. • Of the 30 molecules of ATP formed by the complete combustion of glucose, 26 are formed in oxidative phosphorylation.

• The metabolism of glucose to two molecules of pyruvate in glycolysis yields the remaining four ATP.

• When glucose undergoes fermentation, only two molecules of ATP are generated per glucose molecule.

Respiratory control • Electrons do not flow through the electron‐transport chain unless ADP is available to be converted into ATP.

• The regulation of oxidative phosphorylation by ADP is called acceptor or respiratory control.

• Acceptor control is an example of control of metabolism by energy charge.

Electrons are transferred to O2 only if ADP is concomitantly phosphorylated to ATP. The energy charge regulates the use of fuels.

Electrons do not flow from fuel molecules to O2 unless ATP needs to be synthesized.

The synthesis of ATP from ADP and Pi controls the flow of electrons from NADH and FADH2 to oxygen. The availability of NAD+ and FAD in turn control the rate of the citric acid cycle (CAC). • The mitochondrial protein Inhibitory factor 1 (IF1) inhibits ATP synthase. IF1 may prevent ATP hydrolysis when oxygen is limiting. IF1 specifically inhibits the potential hydrolytic

activity of the F0F1 ATP synthase. • IF1 is overexpressed in some cancers and may facilitate the transition to aerobic glycolysis. This over‐expression plays a role in the induction of the Warburg effect, the switch from oxidative phosphorylation to aerobic glycolysis as the principle means for ATP synthesis.

• If electron transport is uncoupled from ATP synthesis, heat is generated, a process called nonshivering thermogenesis. • Such uncoupling is facilitated in a regulated fashion by uncoupling protein 1 (UCP‐1), also called thermogenin, an intergral protein of the inner mitochondrial membrane. • Uncoupling occurs in mitochondria in brown fat, called brown fat mitochondria. • Pigs have large litters and form nests because they lack UCP‐1. • Adult humans display nonshivering thermogenesis. The action of an uncoupling protein

Uncoupling protein 1 (UCP‐1) also called thermogenin generates heat by permitting the influx of protons into the mitochondria without the synthesis of ATP. UCP-1 generates heat by short-circuiting the mitochondrial proton battery Brown adipose tissue is revealed on exposure to cold.

• The results of PET–CT (positron emission and computerized tomography) scanning show the uptake and distribution of 18F‐ fluorodeoxyglucose (18F‐FDG), a nonmetabolizable glucose analog, in adipose tissue.

• The patterns of 18F‐FDG uptake in the same subject are dramatically different under thermoneutral conditions (left) and after exposure to cold (right). • Inhibition of the electron‐transport chain prevents oxidative phosphorylation by inhibiting the formation of the proton‐motive force.

• Inhibition of ATP synthase by inhibiting proton flow prevents electron transport.

• Uncouplers carry protons across the inner mitochondrial membrane. The electron‐transport chain functions, but ATP synthesis does not occur because the proton gradient can never form.

• Inhibition of the ATP‐ADP translocase prevents oxidative phosphorylation. Sites of action of some inhibitors of electron transport • Mild uncouplers are being sought as a potential treatment for obesity and related disorders.

• One such drug is xanthohumol, an aromatic ketone that is already in use as anti‐cancer drug • Disruption of Complex I is the most common cause of mitochondrial disease.

• Defects in the components of the electron‐transport chain not only reduce ATP synthesis, but increase the amount of reactive oxygen species formed, leading to increased mitochondrial damage. Proton gradients power a wide array of biological processes in all organisms.

Proton gradients are a central interconvertible currency of free energy in biological systems

The proton gradient is an interconvertible form of free energy.