ELECTRON TRANSPORT CHAIN ECDA September 2009

ELECTRON TRANSPORT CHAIN The cells of almost all eukaryotes (animals, plants, fungi, algae, protozoa – in other words, the living things except bacteria) contain intracellular organelles called mitochondria, which produce ATP. Energy sources such as glucose are initially metabolized in the cytoplasm. The products are imported into mitochondria.

ELECTRON TRANSPORT CHAIN Mitochondria continue the process of catabolism using metabolic pathways including the Krebs cycle, fatty acid oxidation, and amino acid oxidation. The end result of these pathways is the production of two kinds of energy-rich electron donors, NADH and FADH2. When metabolized: One NADH molecule = 3 ATP molecules One FADH2 molecule = 2 ATP molecules

ELECTRON TRANSPORT CHAIN Electrons from NADH and FADH2 are passed through an electron transport chain to oxygen, which is reduced to water. This is a multi-step redox process that occurs on the mitochondrial inner membrane.

ELECTRON TRANSPORT CHAIN

ELECTRON TRANSPORT CHAIN Four membrane-bound complexes have been identified in mitochondria. Each is an extremely complex transmembrane structure that is embedded in the inner membrane. Three of them are proton pumps (Complexes I, III, and IV). The structures are electrically connected by lipid-soluble electron carriers and water-soluble electron carriers.

ELECTRON TRANSPORT CHAIN

ETC–Complex I Complex I removes two electrons from NADH and transfers them to a lipid-soluble carrier, ubiquinone (Q) When NADH binds to complex I, it binds to a prosthetic group called flavin mononucleotide (FMN), and is immediately re-oxidized to NAD. FMN then receives the hydrogen from the NADH and two electrons. The reduced FMN form passes the electrons to iron-sulfur clusters that are part of the complex, and forces two protons into the intermembrane space.

ETC–Complex I

ETC–Complex I Electrons pass from complex I to a carrier (Coenzyme Q) embedded by itself in the membrane. From Coenzyme Q electrons are passed to a complex III which is associated with another proton translocation event. Note that the path of electrons is from Complex I to Coenzyme Q to Complex III. Complex II, the succinate dehydrogenase complex, is a separate starting point, and is not a part of the NADH pathway.

ETC–Complex II Complex II (succinate dehydrogenase) is not a proton pump. It serves to funnel additional electrons into the quinone pool (Q) by removing electrons from succinate and transferring them (via FAD) to Q. Complex II consists of four protein subunits: SDHA, SDHB, SDHC, and SDHD. Other electron donors (e.g., fatty acids and glycerol 3- phosphate) also funnel electrons into Q (via FAD), again without producing a proton gradient.

ETC-Complex II

ETC-Complex III Complex III (cytochrome bc1 complex) removes in a stepwise fashion two electrons from QH2 at the QO site and sequentially transfers them to two molecules of cytochrome c, a water-soluble electron carrier located within the intermembrane space. From Complex III the pathway is to cytochrome c then to a Complex IV (cytochrome oxidase complex).

ETC-Complex III

ETC-Complex IV Complex IV (cytochrome c oxidase) removes four electrons from four molecules of cytochrome c and transfers them to molecular oxygen (O2), producing two molecules of water (H2O). Molecular oxygen serves as the final electron sink or acceptor, clearing the way for carriers in the sequence to be reoxidized so that electron transport process can continue

ETC KEY POINTS: Protons are translocated across the membrane, from the matrix to the intermembrane space Electrons are transported along the membrane, through a series of protein carriers Oxygen is the terminal electron acceptor, combining with electrons and H+ ions to produce water As NADH delivers more H+ and electrons into the ETS, the proton gradient increases, with H+ building up outside the inner mitochondrial membrane, and OH- inside the membrane.

ETC INHIBITORS Electron transport chain may be blocked by some compounds known as ETC Inhibitors. ETS inhibitors act by binding somewhere on the electron transport chain, literally preventing electrons from being passed from one carrier to the next. They all act specifically, that is, each inhibitor binds a particular carrier or complex in the ETS. No matter what substrate is used to fuel electron transport, only two entry points into the electron transport system are known to be used by mitochondria: Complexes I and II. o

Two entry points into the ETC system

ETC INHIBITORS

ETC INHIBITORS A consequence of having separate pathways for entry of electrons is that an ETS inhibitor can affect one part of a pathway without interfering with another part. In this case, respiration can still occur depending on choice of substrate. However, some poisons may completely stop ETC and halt respiration.

ETC INHIBITORS Two Mechanism of Inhibition: Irreversible inhibition results in a complete stoppage of respiration via the blocked pathway. Competitive inhibition allows some oxygen consumption since a "trickle" of electrons can still pass through the blocked site. Although it allows some oxygen consumption, competitive inhibition prevents maintenance of a chemiosmotic gradient, thus the addition of ADP can have no effect on respiration.

ETC INHIBITORS An inhibitor may completely block electron transport by irreversibly binding to a binding site. For example, cyanide binds cytochrome oxidase so as to prevent the binding of oxygen. Electron transport is reduced to zero. Breathe all you want - you can't use any of the oxygen you take in. Rotenone, on the other hand, binds competitively, so that a trickle of electron flow is permitted. However, the rate of electron transport is too slow for maintenance of a gradient.

ETC Inhibitors Electron Transport Inhibitors Rotenone Antimycin Cyanide Oligomycin (inhibitor of oxidative phosphorylation)

ETC Inhibitors ROTENONE ANTIMYCIN Used as insecticide Toxic to wildlife, to humans as well as to insects Competitive inhibitor on complexes I and II, thus, blocking respiration ANTIMYCIN Being used in researches binding site for antimycin can be narrowed considerably using combinations of substrates inlcuding succinate, NADH

ETC Inhibitors CYANIDE extremely effective reversible inhibitor of cytochrome oxidase A concentration of 1 mM KCN is sufficient to inhibit oxygen consumption by mitochondria from a vertebrate source by >98%. Cyanide is one of the most deadly compounds in a laboratory.

ATP Synthase Inhibitor OLIGOMYCIN An antibiotic, acts by binding ATP synthase in such a way as to block the proton channel Inhibitor of oxydative phosphorylation it has no direct effect on electron transport or the chemiosmotic gradient

ETC Inhibitors