Chapter 18- Oxidative Phosphorylation

Cellular respiration

Drives ATP formation by transferring electrons to molecular oxygen

Respiratory chain (electron transport chain)

Four large protein complexes that are embedded in the inner mitochondrial membrane

Oxidative phosphorylation

Set of electron-transfer reactions that captures the energy of high-energy electrons from NADH and FADH₂

• takes place in the electron transport chain

• ultimately generates ATP and reduces oxygen to water

• cellular respiration

cellular respiration

The generation of high-transfer potential electrons by the citric acid cycle, their flow through the respiratory chain, and the accompanying synthesis of ATP

Coupling of electron carrier oxidation and ADP phosphorylation

The flow of electrons from reduced carriers such as NADH is highly exergonic

• NADH + 1/2O₂ + H⁺ → H₂O + NAD⁺

  • Favorable

• complexes of the electron-transport chain use released energy to pump protons out of the mitochondrial matrix

  • generates a pH gradient and a transmembrane electron potential that creates a proton-motive force that is used to power the synthesis of ATP

    • ADP + P_i + H⁺ → ATP + H₂O

      • unfavorable

Mitochondria structure

• The citric acid cycle, the electron-transport chain, and ATP synthesis occur in the mitochondria.

• Mitochondria have two membranes (which creates two distinct internal compartments)

  • an outer membrane with porins

  • an extensive, highly folded inner membrane

• intermembrane space (IM space) = compartment between the outer and inner membranes

• matrix = compartment bounded by the inner membrane

Where do most citric acid and fatty acid oxidation reactions take place?

Mitochondrial matrix

Where does Oxidative phosphorylation take place?

Inner mitochondrial membrane

Membranes of mitochondria

• The outer mitochondrial membrane is permeable to most small molecules and ions.

  • because of the presence of mitochondrial porin (voltage- dependent anion channel)

• The inner membrane is folded into structures called cristae that increase the surface area to create more sites for oxidative phosphorylation.

  • impermeable to most ions and polar molecules

  • transporters shuttle metabolites across the membrane

  • has two sides: the matrix side and the cytoplasmic side

What does the respiratory chain consist of?

Four complexes: three proton pumps and a physical link to the citric acid cycle

• electrons flow from NADH to O₂ through three protein complexes embedded in the inner mitochondria membrane

  • NADH-Q oxidoreductase (Complex I)

  • Q-cytochrome c oxidoreductase (Complex III)

  • cytochrome c oxidase (Complex IV)

• electron flow through complexes I, III, and IV is highly exergonic and power generation of a proton gradient

• Complex I, III, and IV are proton pumps

Complex II

Succinate Q-reductase (Complex II) contains succinate dehydrogenase from the citric acid cycle

• electrons from this FADH₂ enter the electron-transport chain at Q-cytochrome c oxidoreductase

• It does not pump protons

Pathway of electrons through the complexes

Complexes I, III, and IV appear to be associated in a supramolecular complex

• facilitates the rapid transfer of substrate

• Prevents the release of reaction intermediates

Coenzyme Q

hydrophobic quinone that diffuses rapidly within the inner mitochondrial membrane

• can exist in several oxidation states

• couple electron-transfer reactions to proton binding and release

• functions as an electron carriers

  • the reduced form carries electrons from Complex I to Complex III

  • It transfers electrons from FADH₂ from the citric acid cycle to Complex III

Cytochrome c

small soluble protein that is loosely associated with the inner mitochondrial membrane

• shuttles electrons from Complex III to Complex IV

• present in all organisms with mitochondrial respiratory chains

• highly conserved across species

• cytochrome c from any eukaryotic species will react in vitro with the cytochrome c oxidase from any other species

cytochromes

electron-transferring proteins that contain a heme prosthetic group

Iron-sulfur clusters

• common components of the electron-transport chain

• They are in every complex and transfer electrons

NADH-Q oxidoreductase (Complex I)

Proton pump that serves as the entry point for electrons from NADH

• encoded by genes in the mitochondria and nucleus

• Complex I catalyzes the reaction:

  • NADH + Q + 5H⁺ → NAD⁺ + QH₂ + 4H⁺_{intermediate space}

• high potential electrons of NADH enter the respiratory chain at NADH-Q oxidoreductase

How many oxidation states does flavin have?

Two

• NADH binds and the transfer of its two high-potential electrons to the flavin mononucleotide (FMN) prosthetic group of Complex I

  • yields the reduced form, FMNH₂

  • electrons are then passed to a series of iron-sulfur proteins clusters in Complex I

Q Chamber

enclosed site where Q accepts electrons from NADH

Electron transfer through NADH-Q Oxidoreductase is coupled to…

Proton transfer reactions

How does structural cooperation pump protons out of the matrix?

• Q accepts two electrons from NADH, generating Q^2-

• Q^2- negative charge causes a conformational change in LH and βH elements, leading to a change in the structures of the connected vertical helices that change the pKa of the amino acids

• H+ from the matrix binds to amino acids, dissociates into a water-lined channel, and enters the intermembrane space.

• The flow of two electrons from NADH to coenzyme Q through Complex I leads to the pumping of 4 H+ out of the matrix of the mitochondrion.

Reduction of Q^2- to QH₂

• Q^2- takes up two protons from the matrix as it is reduced to QH₂

  • contributes to the formation

• QH₂ subsequently leaves the enzyme for the Q pool, allowing another reaction cycle to occur

Succinate Q-reductase (Complex II)

Integral membrane protein complex of the inner mitochondrial membrane

• contains succinate dehydrogenase

• does not pump protons, reusltin in less ATP being formed in the oxidation of FADH₂ than NADH

What is the entry point for electrons from FADH₂ of flavoproteins

Ubiquinol

Complex II

Electrons from FADH₂ (generated in the citric acid cycle) are transferred to Fe-S Centers and then to Q to form QH₂

Electon flow from ubiquinol to cyctochrome c through Q-cytochrome c oxidoreductase

• electrons from QH₂ are passed to cytochrome c (Cyt c) by Q-cytochrome c oxidoreductase (Complex III)

  • leads to the net transport of 2 H⁺ to the intermembrane space

  • QH₂ + 2 Cyt c_ox + 2H⁺_matrix → Q + 2 Cyt c_red + 4H⁺_{intermembrane space}

• Complex II contains:

  • two types of cytochromes names b and c₁

  • four prosthetic group: three hemes and a 2F3-2S cluster

cytochrome c oxidase (Complex IV)

Catalyzes the transfer of four electrons from four reduced molecules of cytochrome c to O₂

How does cytochrome c oxidase catalyze the reduction of molecular oxygen to water?

• Four H+ are used to reduce O2 to H2O.

• Four H+ are pumped into the intermembrane space.

• ∆G°′ captured in the form of a proton gradient for ATP synthesis

Steps of Cytochrome c catalyzing reaction to reduce oxygen to water

• Step 1: electrons from two reduced cytochrome c molecules flow to CuA/CuA, to heme a, to heme a3, to CuB

  • one stops at heme a3

  • one stops at CuB

• Step 2: reduced heme a3 and CuB bind O2, forming a peroxide bridge between them

• Step 3: electrons from two more cytochrome c molecules and H+ from the matrix bind to each oxygen, cleaving the peroxide bridge

• Step 4: reactions with two more H+ releases two molecules of H2O and resets the enzyme

Mechanism of cytochrome c oxidase prevents early oxygen release

Two components of proton transport by cytochromse c oxidase

• Four chemical protons reduce O2 to two H2O.

• Cytochrome c oxidase uses free energy from this reduction to pump 4 H+ from the matrix into the intermembrane space

Electrons flow via two pathways through the electron-transport chain

Respirasome

A massive complex in humans consisting of two copies of Complex I, Complex III, and Complex IV

• Structure allows for Complex II to associate in a gaph between Complexes I and IV

• enhances efficiency

• Most of the electron-transport chain is organized into the respirasome

How are toxic reactive oxygen species limited in the mitochondria?

• scavenged by protective enzymes

  • partial reduction of O₂ generates highly reactive oxygen derivatives called reactive oxygen species (ROS)

  • ROS are implicated in aging and a growing list of diseases

  • ROS include superoxide ion, peroxide ion, and hydroxyl radical

  • cytochome c oxidase does not release ROS by holding O₂ tightly between Fe and Cu ions

• Superoxide dismutase (SOD) = enzyme that scavenges superoxide racidals by catalyzing the conversion of two radical into hydrogen peroxide and molecular oxygen

  • Eukaryotes contain two forms of SOD:

    • a manganese-containg version located in mitochondria

    • a copper-and-zinc-dependent cytoplasmic form

  • exercise is associated with increased SOD expression

Two phases of superoxide dismutase mechanism

Catalase

A ubiquitous heme protein that catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen

What powers the synthesis of ATP?

A proton gradient

• flow of NADH to O₂ is an exergonic process

• Synthesis of ATP is an endergonic process

ATP synthase (Complex V)

A molecular assembly in the inner mitochondrial membrane that carries out the synthesis of ATP

Chemiosmotic hypothesis

Proposes that electron transport and ATP syntheis are ciupled by a proton gradient across the inner mitochondrial membrane

• suggested that ATP ormation is powered by a proton gradient

Proton-Motive Force

The energy-rich unequal distribution of protons across a membrane

• consists of a chemical gradient and a change gradient

• powers the synthesis of ATP

• proton-motive force (delta p) = chemical gradient (delta pH) + charge gradient

ATP synthase composition

Composed of a proton-conducting unit and a catalytic unit

Made up of two components resembling a ball on a stick:

• The F₀ (stick) component is embedded in the inner mitochondrial membrane and contains the proton channel

• The F₁ (ball) component protrudes into the mitochondrial matrix and contains the catalytic activity

Mitochondria ATP synthase forms homodimers

• stabilizes the individual enzymes to the rotational forces required for catalysis

• Facilitates the survature of the inner mitochondrial membrane

How does ATP Synthase assist in the formation of cristae?

Cristae formation allows proton pumps to localize the proton gradient in the civinity of the synthases, which are located at the tips of the cristae

• enhances efficiency of ATP synthesis

Proton flow through ATP synthase

• leads to the release of tightly bound ATP via the binding-change mechanism

• ATP synthase catalyzes the formation of ATP from ADP and P_i

• Actual substrates are ATP and ADP complexed with Mg²⁺

• A terminal oxygen atom of ADP attacks the phosphorus atom of P_i

  • form a pentacovalent intermediate that dissociates into ATP and H₂O

• allows for the release of the newly symthesized ATP

• the F₁ subunit of the ATPase contains three β subunits

  • There are three active sites on the enzyme

• Binding-change mechanism states a β subunit can
  perform three sequential steps in ATP synthesis by
  changing conformation:

  • Step 1: ADP and Pi binding

  • Step 2: ATP synthesis

  • Step 3: ATP release

    
    

What is the ATP yield from each complete turn of the motor?

Each 360-degree rotation of the γ subunit leads to the synthesis and release of three molecules of ATP.

• protons must travel around the c Ring to Cross the membrane

Oxidative phosphorylation overview

How does NADH shuttle its protons to the mitochondria?

• The respiratory chain regenerates NAD+ for use in glycolysis, but the inner mitochondrial membrane is impermeable to NADH and NAD+.

• glycerol 3-phosphate shuttle = one means of transporting electrons from NADH into the electron transport chain

• When cytoplasmic NADH transported by this shuttle is oxidized by the respiratory chain, 1.5 ATP are formed

  • because FAD rather than NAD+ is the electron acceptor

Glycerol 3-phosphate shuttle steps

Step 1: Electrons from NADH are transferred to dihydroxyacetone phosphate to form glycerol 3-phosphate.
Step 2: Glycerol 3-phosphate is moved into the mitochondrion and reoxidized to dihydroxyacetone phosphate.

• Electrons pass to an FAD prosthetic group, forming FADH2.

Step 3: Reduced flavin transfers its electrons to a molecule of Q, which enters the respiratory chain as QH2.

Allows for rapid rates for oxidative phosphorylation at the cost of energetic efficiency

malate-aspartate shuttle

Transports electrons from cytoplasmic NADH into mitochondria

• forms mitochondrial NADH

• in the heart and liver

• mediated by two membrane carriers and four enzymes

Preserves all the chemical potential energy of cytoplasmically generated NADH

Malate-aspartate shuttle steps

Step 1: Electrons are transferred from NADH in the cytoplasm to oxaloacetate, forming malate and NAD+.
Step 2: malate traverses the inner mitochondrial membrane in exchange for α-ketoglutarate.
Step 3: In the matrix, malate is then reoxidized by NAD+, forming oxaloacetate and NADH to form NADH
Step 4: Glutamate donates an amino group to oxaloacetate, forming aspartate and α-ketoglutarate.
Step 5: Aspartate and α-ketoglutarate enter the cytoplasm.
Step 6: In the cytoplasm, oxaloacetate is regenerated, and the cycle is restarted.

ATP-ADP translocase (adenine nucleotide translocase, ANT)

Specific transport protein that enables the exchange of cytoplasmic ADP for mitochondrial ATP

• constitutes 15% of the protein of the inner mitochondrial membrane

ATP and ADP bind to ANT without Mg²⁺

Inhibition of ANT leads to the inhibition of cellular respiration

Entry of ADP into mitochondria is coupled to the exit of ATP by ATP-ADP translocase

ATP-ADP translocase catalyzes the exchange of entry of ADP and ATP

• the translocase contains a single nucleotide-binding site that alternately faces the matrix and the cytoplasmic sides of the membrane

Phosphate Carrier

Mediates the exchange of cytoplasmic H₂PO^4- for mitochondrial OH^-

• Works in concert with ANT to exchange cytoplasmic ADP and P_i for matrix ATP

ATP synthasome

a large complex composed of ATP synthase and the two transporters

Regulation of cellular respiration

• The ATP needs of the cell determine the rate of the respiratory pathways and their components

• Approximately 30 molecules of ATP formed when glucose is completely oxidized to CO₂

  • 26 are formed in oxidative phosphorylation

  • 2 are formed in the citric acid cycle

  • 2 are formed in glycolysis

• Electrons do not flow through the electron-transport
  chain unless ADP is available to be converted into ATP

• The regulation of the rate of oxidative phosphorylation by ADP level is called respiratory (or acceptor) control.

• At low ADP levels:

  • NADH and FADH2 are not consumed by the electron- transport chain.

  • the citric acid cycle slows because there is less NAD+ and FAD to feed the cycle.

Regulation of ATP synthase

• inhibitory factor 1 (IF1) = an evolutionarily conserved mitochondrial protein that specifically inhibits the potential hydrolytic activity of ATP synthase

  • dimerizes when matrix pH decreases

  • dimers bind tightly to ATP synthase dimers, preventing the β subunits from changing conformation

  • essential in cases where tissues may be O2 deprived (e.g., stroke or heart attack) and the electron-transport chain cannot generate the proton-motive force

Regulated uncoupling

Leads to the generation of heat

• nonshivering thermogenesis = the ability to generate heat without using shivering by uncoupling oxidative phosphorylation from ATP synthesis

  • occurs in mitochondria-rich brown adipose tissue in animals

  • activated in response to a drop in the core body temperature

• uncoupling protein 1 (UCP-1; also called thermogenin) = an inner mitochondria membrane protein that transports protons from the intermembrane space to the matrix with the assistance of fatty acids

  • generates heat by transporting protons without the synthesis of ATP

  • energy of the proton gradient, normally captured as ATP is released as heat as the protons flow through UCP-1 to the mitochondrial matrix

• UCP-2= uncoupling protein found in a wide variety of
  tissues

• UCP-3 = uncoupling protein found in skeletal muscle and
  brown fat

• UCP-2 and UCP-3:

  • share >50% identity with UCP-1

  • may play a role in energy homeostasis

  • may be important in regulating body weight

• Obesity leads to a decrease in brown adipose tissue.

Power transmission by proton gradients

• central motif of bioenergetics

• Proton gradients power a variety of energy-requiring processes

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