b 12 revisited. oxidation of propionyl-coa most dietary fatty acids are even-numbered many plants...

B12 revisited

Oxidation of Propionyl-CoA

• Most dietary fatty acids are even-numbered

• Many plants and some marine organisms also synthesize odd-numbered fatty acids

• Propionyl-CoA forms from -oxidation of odd-numbered fatty acids

• Bacterial metabolism in the rumen of ruminants also produces propionyl-CoA

Figure 25-20 The rearrangement catalyzed by methylmalonyl-CoA mutase.

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Figure 25-21Structure of

5’-deoxyadenosyl-cobalamin

(coenzyme B12).

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Homolytic cleavageEach product gets 1 electron from the bond

Cobalt acts as a reversible free radical generator!

Adenosyl radical abstracts H from substrate

Oxidative Phosphorylation

• Coupling of reduction of O2 with ATP production– Substrate level phosphorylation?– High energy intermediate structure/state?– Something else?

Figure 22-12 Electron micrographs of mouse liver mitochondria. (a) In the actively respiring state. (b) In the resting state.

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Chemiosmotic Theory

• How to make an unfavorable

ADP + Pi = ATP

possible?• Phosphorylation of ADP is not a result of a direct

reaction between ADP and some high energy phosphate carrier

• Energy needed to phosphorylate ADP is provided by the flow of protons down the electrochemical gradient

• The electrochemical gradient is established by transporting protons against the electrochemical gradient during the electron transport

Chemiosmotic Energy Coupling Requires Membranes

• The proton gradient needed for ATP synthesis can be stably established across a topologically closed membrane– Plasma membrane in bacteria– Cristae membrane in mitochondria– Thylakoid membrane in chloroplasts

• Membrane must contain proteins that couple the “downhill” flow of electrons in the electron transfer chain with the “uphill” flow of protons across the membrane

• Membrane must contain a protein that couples the “downhill” flow of proton to the phosphorylation of ADP

Figure 22-3 Freeze-fracture and freeze-etch electron micrographs of the inner and outer

mitochondrial membranes.

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How could you identify and reconstruct the ETC?

• Intact mitochondria• Submitochondrial particles

• Identify components:

– Pyridine-linked DH– Flavin-linked DH– Iron-sulfur proteins– Cytochromes– Ubiquinone

Coenzyme Q or Ubiquinone

• Ubiquinone is a lipid-soluble conjugated dicarbonyl compound that readily accepts electrons

• Upon accepting two electrons, it picks up two protons to give an alcohol, ubiquinol

• Ubiquinol can freely diffuse in the membrane, carrying electrons with protons from one side of the membrane to another side

How do they fit together?

• Redox potentials

• Visualize redox by UV/vis

• INHIBITORS!!!

Cytochrome c Absorbs Visible Light

• Intense Soret band near 400 nm absorbs blue light and gives cytochrome c an intense red color

• Cytochromes are sometimes named by the position of their longest-wavelength peak

Iron-Sulfur Centers

• Found in several proteins of electron transport chain, including NADH:ubiquinone oxidoreductase

• Transfers one electron at a time

Figure 22-9 The mitochondrial electron-transport chain.P

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In the presence of antimycin A and an electron donor, is Cyt b in its oxidized or reduced state?

Separation of functional complexes of the respiratory chain.

Figure 22-14The mitochondrial electron-transport chain.P

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Path of electrons from NADH, succinate, fatty acyl–CoA, and glycerol 3-phosphate to ubiquinone

NADH:Ubiquinone Oxidoreductasea.k.a. Complex I

• One of the largest macro-molecular assemblies in the mammalian cell

• Over 40 different polypeptide chains, encoded by both nuclear and mitochondrial genes

• NADH binding site in the matrix side• Non-covalently bound flavin mononucleotide

(FMN) accepts two electrons from NADH• Several iron-sulfur centers pass one electron at

the time toward the ubiquinone binding site

NADH:ubiquinone oxidoreductase (Complex I).

Structure of NADH:Ubiquinone Oxidoreducase

The complete macromolecular assembly can be seen in electron microscopy. Part of the bacterial protein has been crystallized but the 3D structure of the membrane-spanning domain remains unknown

NADH:Ubiquinone Oxidoreducase is a Proton Pump

• Transfer of two electrons from NADH to uniquinone is accompanied by a transfer of protons from the matrix (N) to the inter-membrane space (P)

• Experiments suggest that about four protons are transported per one NADH

NADH + Q + 5H+N = NAD+ + QH2 + 4 H+

P

• Reduced coenzyme Q picks up two protons

• Despite 50 years of study, it is still unknown how the four other protons are transported across the membrane

Succinate Dehydrogenasea.k.a. Complex II

• FAD accepts two electrons from succinate

• Electrons are passed, one at a time, via iron-sulfur centers to ubiquinone that becomes reduced QH2

Structure of Complex II (succinate dehydrogenase).

= path of e- transfer

Heme b protects against rogue electrons forming reactive oxygen species

Cytochrome bc1 Complexa.k.a. Complex III

• Uses two electrons from QH2 to reduce two molecules of cytochrome c

Cytochrome bc1 complex (Complex III).

a dimer of identical monomers, each with 11 different subunits.

Complex has two distinct binding sites for ubiquinone, QN and QP. The interface between monomers forms two caverns, each containing a QP site from one monomer and a QN site from the other. The ubiquinone intermediates move within these sheltered caverns.

The Q Cycle

• Experimentally, four protons are transported across the membrane per two electrons that reach CytC

• Two of the four protons come from QH2

• The Q cycle provides a good (but complicated) model that explains how two additional protons are picked up from the matrix

Animation of Q cycle

• http://www.life.illinois.edu/crofts/qcycle_model.html

• http://www.macromol.uni-osnabrueck.de/BC1_complex.php

The Q cycle, shown in two stages

Cytochrome c

• Cytochrome c is a soluble heme-containing protein in the intermembrane space

• Heme iron can be either ferrous(Fe3+, oxidized) or ferric(Fe2+, reduced)

• Cytochrome c carries a single electron from the cytochrome bc1 complex to cytochrome oxidase

Cytochrome Oxidase a.k.a. Complex IV

• Mammalian cytochrome oxidase is a membrane protein with 13 subunits

• Contains two heme groups

• Contains copper ions

– Two ions (CuA) form a binuclear center

– Another ion (CuB) bonded to heme forms Fe-

Cu center

Cytochrome Oxidase Passes Electrons to O2

• Four electrons are used to reduce one oxygen molecule into two water molecules

• Four protons are picked up from the matrix in this process

• Four additional protons are passed from the matrix to the inter-membrane space by an unknown mechanism

Summary of the Electron Flow in the Respiratory Chain

Proton-motive Force

• The proteins in the electron transport chain created the electrochemical proton gradient by one of the three means:

–actively transported protons across the membrane via poorly understood mechanisms

–passed electrons to coenzyme Q that picked up protons from the matrix

–took electrons from QH2 and released the

protons to the inter-membrane side

The inner mitochondrial membrane separates two compartments of different [H+], resulting in differences in chemical concentration (ΔpH) and charge distribution (Δψ) across the membrane.

Chemiosmotic Model for ATP Synthesis

• Electron transport sets up a proton-motive force

• Energy of proton-motive force drives synthesis of

ATP

Energy Calculator

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Mitochondrial ATP Synthase Complex

• The proton-motive force causes rotation of the central shaft

• This causes a conformational change within all the three pairs

• The conformational change in one of the three pairs promotes condensation of ADP and Pi into

ATP

Figure 22-43 Model of the E. coli F1F0–ATPase.

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Rotational Catalysis

Movies

• http://atom.chem.wwu.edu/sacahill/472/atp%20synthase.mov

• http://atom.chem.wwu.edu/sacahill/472/atp%20synthase2.mov

• http://atom.chem.wwu.edu/sacahill/472/rotarymech.mov

Figure 22-46 Uncoupling of oxidative phosphorylation.

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Figure 22-47 Mechanism of hormonally induced uncoupling of oxidative

phosphorylation in brown fat mitochondria.

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ATP Yield From Glucose

Let’s Sing!

• Lyrics?

Light Energy is Converted to ATP in Plant Chloroplasts

Various Pigments Harvest the Light Energy

The energy is transferred to the photosynthetic reaction center

Light-Induced Redox Reactions and Electron

Transfer Cause Acidification of Lumen

The proton-motive force across the thylakoid membrane drives the synthesis of ATP

Flow of Protons: Mitochondria, Chloroplasts, Bacteria

• According to endosymbiotic theory, mitochondria and chloroplasts arose from entrapped bacteria

• Bacterial cytosol became mitochondrial matrix and chloroplast stroma