Metabolic modes of energy generation

• Respiration – couple substrate oxidation to the ultimate reduction of an extrinsic chemical such as O2, DMSO, etc.

• Fermentation – couple substrate oxidation to reduction of internally generated substrates

• Photosynthesis – harvest light energy to facilitate electron transport in energy generating mechanism

Chemiosmotic Theory

• The transmembrane differences in proton concentration are the reservoir for energy extracted from biological oxidation reactions

- Peter Mitchell

Fermentation utilizes substrate level phosphorylation, more on thatlater

Oxidative phosphorylation and photophosphorylation

have similarities and differences • In eukaryotes, both are organellar processes;

mitochondria and chloroplast

• Both involve flow of electrons through membrane components

• Free energy from electron flow is used to pump protons across a membrane

• Flow of protons back drives ATP synthesis

• In aerobic systems, Oxidative phospho. Reduces O2 to H2O, while photophospo. Can oxidize H2O to O2

• Oxidative energy

generation leads to ATP,

water, and oxidized electron

carriers

Eukaryotic cells

Oxidative phosphorylation is a mitochondrial process

• Although the mitochondria

imports some biomolecules

from the cytoplasm, it contains

Citric acid cycle enzymes, it’s own

genome, etc. Notice the cristae

which increase membrane surface

area.

Electron carriers initiate oxidative phosphorylation

• Pools of electrons linked to carriers such as NAD, NADP, FMN, and FAD are generated by catabolic mechanisms (mostly NADH is generated)

• Note when depicted as NAD+, the intent is to reflect oxidation state, NOT charge on the molecule

Electrons are passed to membrane components

• For instance, NADH is oxidized by a membrane bound enzyme NADH dehydrogenase, which subsequently passes electrons to quinones, and so on.

• Various steps are linked to proton translocation out of the mitochondrial inner membrane

A membrane is a prerequisite for biological energy generation

This serves as an impermeable barrier to many solutessuch as H+, even H2O. Polar molecules cannot traversethe hydrophobic layer.

Cells can alter the fatty acid content of their membranes

• Sterols

modify

fluidity

also

Ubiquinone (CoQ) is a lipid soluble two electron, two proton

carrier• Plants –

Plastoquinone

• Bacteria –

menaquinone

Freely diffusible in

Lipid bilayer

Cytochromes classified on basis of porphyrin ring

Another example of a spectroscopic bioassay

Iron-sulfur proteins carry electrons and do more…

• At least eight iron-sulfur proteins act in mitochondrial electron transfer

• Also Fe-S centers have been shown to be sensors of aerobic/anaerobic gene expression

Determining the order of electron transfer

• Standard reduction potentials

• Spectroscopic measure of carrier oxidation

• Inhibitors– Rotenone inhibits NADH dehydrogenase– Antimycin A inhibits cytochrome b– Cyanide, azide, or Carbon monoxide inhibit

cytochrome oxidase aa3

Experimental evidence for electron transfer order

The order of electron transfer under aerobic conditions

Cytosolic-derived NADH must be shuttled into the mitochondria• Although citric acid cycle and fatty acid

oxidation occur in the “right” place (mitochondrial matrix), glycolysis is cytoplasmic and NADH from this pathway must be shuttled into the matrix of the mitochondria (membrane is impermeable to this compound; no transporter)– Glycerol-3-phosphate shuttle– Malate-Aspartate shuttle

Glycerol-3-phosphate shuttle

• 2 e- fromNADH to

FADH2

Get only2 ATP from

FADH2 vs3 from NADHMore on that later

Malate-Aspartate Shuttle

General class of transporters

Electron transport is accomplished by enzyme complexes

NADH:ubiquinone oxidoreductase utilizes NADH generated from catabolic reactions

• This is a huge protein complex ~900,000 kDa

• Electron transfer from NADH to ubiquinone is coupled to the translocation of protons through the protein, with a stoichiometry of 2H+/e-– NADH + H+ + Q NAD+ + QH2

An electron’s path through this complex

• Oxidation of NADH transfers two electrons to FMN bound to the enzyme, and releases a proton into the matrix

• The electrons are passed from FMN through a series of Fe-S centers the last one being called N-2 (Six in the case of the mitochondrial enzyme, but only four appear to be universally conserved)

• N-2 reduces ubiquinone

Proton pumping by this complex

• Experiments suggest one proton is pumped into the intermembrane space during NADH to N-2 transfer of one electron, and a second proton during N-2 to ubiquinone transfer of one electron

• Recall the stoichiometry, 2 protons per electron – this means four protons in total are pumped for each NADH oxidation