OXIDATIVE PHOSPHORYLATION

Oxidative Phosphorylation  The process in which ATP is formed as a result of the transfer of electrons from NADH or FADH 2 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?

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

Succinate – Q reductase  Composed of 4 subunits  Prosthetic groups: FAD and Fe-S  No transport of protons for enzymes that transport electrons from FADH 2. Hence, less ATP is produced for the oxidation of FADH 2

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 QH 2 to oxidized cytochrome c and concommitantly pump protons out of the mitochondrial matrix

Q – Cytochrome c oxidoreductase (Cytochrome bc 1 complex)

Cytochrome bc 1 complex  A dimer with each monomer containing 11 subunits  Contains 3 hemes  2 b-types (b H and b L )  1 c-type  The enzyme also contains Rieske center  It also has 2 binding sites : Q 0 and Q i  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 (Cu A /Cu A ) and B (Cu B )  heme A (yellow) is composed of heme a and heme a3  Cu A (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 Cu A /Cu A  Heme a3 passes electrons to Cu B  Heme a3 and Cu B form the active center at which the oxygen is reduced to water

Cytochrome c oxidase mechanism

ATP synthesis ΔG˚’ = kcal / mol ΔG˚’ = +7.3 kcal / mol

ATP synthase  Membrane embedded enzyme  2 subunits: F 1 and F o  F 1 : 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