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OXIDATIVE PHOSPHORYLATION
Oxidative Phosphorylation
The process in which ATP is formed as a result of the transfer of electrons from NADH or FADH2 to oxygen by a series of electron carriers
Takes place in the mitochondria
Electron flow proton flow pH gradient and transmembrane electrical potential proton motive force
Mitochondria
2 µm in length; 0.5 µm in diameter
Outer membrane is permeable to small molecules and ions because of the porins (VDAC)
Inner membrane impermeable
2 faces: matrix (neg) cytosol (pos)
REDOX CONCEPTS
A strong reducing agent donates electrons and has negative reduction potential while a strong oxidizing agent accepts electrons and has positive reduction potential
Standard reduction potential (Eo) How much energy will be produced from
the reduction of oxygen with NADH?
''
EnFG
Electron carriers
Flavins Iron-sulfur clusters Quinones Hemes Copper ions
Flavins
The isoalloxazine ring can undergo reversible reduction accepting either 1 or 2 electrons in the form of either 1 or 2 hydrogen atoms
Variability in standard reduction potential is also an important feature
Iron – Sulfur Clusters
Iron – Sulfur Proteins
Iron is not present in the heme but in association with inorganic sulfur atoms or the sulfur of cysteine.
Rieske iron-sulfur proteins are a variation in which 1 iron atom is coordinated with 2 His residues
All iron-sulfur proteins participate in 1 electron transfer
There are at least 8 Fe-S clusters in the respiratory chain
Quinones
Ubiquinone or Coenzyme Q
Can accept 1 or 2 electrons
Can act at the junction between 2-electron donor and 1-electron acceptor because it is freely diffusable
Plays a central role in coupling electron flow and proton movement because it carries both electrons and protons
Hemes (cytochromes)
Hemes (cytochrome)
3 classes: a, b, c (difference in light absorption spectra)
Of the three, the heme of cytochrome c is covalently bonded to the protein
The standard reduction potential of the hemes depends on its interaction with the protein side chains
The Four Complexes of the Respiratory Chain
NADH – Q oxidoreductase (Complex I) Succinate – Q reductase (Complex II) Q – cytochrome c oxidoreductase
(Complex III) Cytochrome c oxidase (Complex IV)
NADH – Q oxidoreductase
Aka NADH dehydrogenase MW: 880 kDa Consists of at least 34 polypeptide
chains Prosthtic groups: FMN and Fe-S
clusters Catalyzes 2 simultaneous and
obligately coupled processes
NADH-Q oxidoreductase
NADH – Q oxidoreductase
1. Exergonic transfer to ubiquinone of a hydride ion from NADH and a proton from the matrix
2. Endergonic transfer of four protons from the matrix to the intermembrane space
PN HQHNADQHNADH 45 2
Succinate – Q reductase
Composed of 4 subunits
Prosthetic groups: FAD and Fe-S
No transport of protons for enzymes that transport electrons from FADH2. Hence, less ATP is produced for the oxidation of FADH2
Cytochrome
An electron transferring protein that contains a heme prosthetic group
The iron alternates between reduced and oxidized forms during electron transport
Q- cytochrome c oxidoreductase catalyzes the transfer of electrons from QH2 to oxidized cytochrome c and concommitantly pump protons out of the mitochondrial matrix
Q – Cytochrome c oxidoreductase (Cytochrome bc1 complex)
Cytochrome bc1 complex
A dimer with each monomer containing 11 subunits
Contains 3 hemes 2 b-types (bH and bL) 1 c-type
The enzyme also contains Rieske center It also has 2 binding sites : Q0 and Qi Q -cycle
Q - cycle
Cytochrome c oxidase
Catalyzes the reduction of molecular oxygen to water
Oxidation of the reduced Cyt c generated in complex III w/c is coupled w/ reduction of oxygen to 2 molecules of water
Cytochrome c oxidase
The enzyme contains 2 heme A groups and 3 copper ions arranged as 2 copper centers, A (CuA/CuA ) and B (CuB)
heme A (yellow) is composed of heme a and heme a3
CuA (blue) contains 2 copper ions linked by bridging cysteine residues
Cytochrome c oxidase
Heme a and a3 are located in different environments within the enzyme
Heme a carries electrons from CuA
/CuA Heme a3 passes electrons to CuB Heme a3 and CuB form the active
center at which the oxygen is reduced to water
Cytochrome c oxidase mechanism
ATP synthesis
NADOHHONADH 222
1
OHATPHPADP i 2
ΔG˚’ = -52.6 kcal / mol
ΔG˚’ = +7.3 kcal / mol
ATP synthase
Membrane embedded enzyme 2 subunits: F 1 and Fo
F1 : protrudes from the mitochondrial matrix and contains the catalytic activity
: α 3 β 3 γ δ ε
: alpha and beta units are arranged hexamerically : beta subunit participates in catalysis
: gamma subunit breaks the symmetry of the alpha and beta hexamer .
ATP synthase
Fo : hydrophobic segment that spans the inner mitochondrial membrane
: contains the proton channel of the complex
: consists of a ring comprising 10 – 14 c subunits
embedded in the membrane
: a single a subunit binds outside the ring
* The role of the proton gradient is not to form ATP but to release it from the synthase
Binding –Change Mechanism
The changes in the properties of the three β subunits allows sequential ADP and Pi binding, ATP synthesis and ATP release
Three conformations for the β subunit: T (tight) – binds ATP with great avidity but cannot
release the ATP L (loose) – bind ADP and Pi but cannot release ADP and
Pi O (open) – can exist with a bound nucleotide like T and
L but it can also convert to form a more open conformation and release bound molecules
The interconvertion of these three forms can be driven by the rotation of the γ subunit
Proton flow around the c ring
The mechanism depends on the structures of a and c subunit of Fo
Each polypeptide chain forms a pair of α –helices that span the membrane
An aspartic acid (Asp61) is found in the middle of the second helix
The a subunit consists of two proton half channels that do not span the membrane
The a subunit directly abuts the ring comprising the c subunits , with each half channel directly interacting with one c subunit
a and c subunits of Fo
INHIBITORS OF THE ETC
Rotenone - blocks complex I Amytal – blocks complex I Antimycin A – blocks complex III Cyanide – blocks complex IV