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?

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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