B 12 revisited
Dec 19, 2015
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.
Pag
e 92
3
Figure 25-21Structure of
5’-deoxyadenosyl-cobalamin
(coenzyme B12).
Pag
e 92
3
Pag
e 92
6Proposed mechanism of methylmalonyl-CoA mutase.
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.
Pag
e 80
6
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.
Pag
e 79
9
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
age
803
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
age
808
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
http://bcs.whfreeman.com/lehninger5e/pages/bcs-main.asp?v=&s=19000&n=00040&i=19040.01&o=|00610|00580|00590|00510|00540|00600|00550|00570|00630|00010|00020|00030|00040|00070|00080|00090|00100|01000|02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|14000|15000|16000|17000|18000|19000|20000|21000|22000|23000|24000|25000|26000|27000|
28000|99000|
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.
Pag
e 83
2
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.
Pag
e 83
4
Figure 22-47 Mechanism of hormonally induced uncoupling of oxidative
phosphorylation in brown fat mitochondria.
Pag
e 83
5
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