Respiratory chain components


The system of mitochondrial enzymes and redox carrier molecules which ferry reducing equivalents from substrates to oxygen are collectively known as the electron transport system, or the respiratory chain. This system captures the free energy available from substrate oxidation so that it may later be applied to the synthesis of ATP. Many respiratory chain components were first identified in crude homogenates through their spectral properties, which frequently change when a carrier is oxidised or reduced. Fractionation of mitochondria in the presence of mild detergents or chaotropic salts dissected the respiratory chain into four large multi-subunit complexes containing the principal respiratory carriers, named Complex 1 to Complex 4. These substantial protein "icebergs" float in the sheet of inner membrane lipids, often presenting one face to the mitochondrial matrix and another to the inter - membrane space. Many of their components have now been isolated in a relatively pure form. Other membrane bound enzymes such as the energy linked transhydrogenase (ELTH) are also present which fulfil ancillary roles.

The main components participate in the approximate order of their redox potentials, and the bulky complexes are linked by low molecular weight mobile carriers which ferry the reducing equivalents from one complex to the next. Except for succinate dehydrogenase (complex 2) all these complexes pump protons from the matrix space into the cytosol as they transfer reducing equivalents (either hydrogen atoms or electrons) from one carrier to the next. The diagram above shows the flow of reducing equivalents in purple, and movement of the positively charged protons in red. Proton pumping is an arduous task which creates substantial pH and electrical gradients across the mitochondrial inner membrane. These protons eventually re-enter the matrix space via the F1 ATPase, driving the synthesis of ATP as they return.

The number of protons and the number of positive charges crossing the inner membrane need not necessarily agree for each individual transmembrane protein, although the accounts must balance for the whole ensemble. This discrepancy is illustrated on the diagram above, and is explained in greater detail below.

The sequence of of the carriers in the respiratory chain was clarified by the use of specific inhibitors to block the flow of electrons from substrates to oxygen. All components on the substrate side of a block become more reduced, while those which follow become more oxidised. These changes can often be observed spectroscopically. If a range of inhibitors are available, acting at different places, then their precise points of action and the order of the carriers is defined.

Further information was gleaned from the use of artificial redox mediators. Electron donors, such as ascorbate [vitamin C] plus the synthetic compound tetramethyl phenylene diamine can contribute electrons to cytochrome c, while artificial acceptors such as ferricyanide would collect electrons from complex 1 or complex 2 and by-pass the remainder of the chain. This could be particularly informative when used in conjunction with specific inhibitors, or when ATP yields were measured for particular segments of the chain.

Respiratory Chain Components
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Location Prosthetic     
groups
Function 
NADPH / NADP (almost 100% in the reduced form) matrix space (separate cytosolic pool) - mobile carrier
energy-linked transhydrogenase NADPH + NAD+
=> NADH + NADP+
membrane spanning protein none proton pump 2H+/2e-1
NADH / NAD (less than 30% in the reduced form) matrix space (separate cytosolic pool) - mobile carrier
NADH dehydrogenase
(complex 1)
membrane spanning multi-subunit protein non-heme iron
& FMN
proton pump 4H+/2e-1
succinate dehydrogenase
(complex 2)   [see note 1]
membrane spanning multi-subunit protein non-heme iron
& FAD
no proton pumping
ubiquinol / ubiquinone dissolved in the inner membrane lipids - mobile carrier
ubiquinol:cytochrome c reductase (complex 3) membrane spanning multi-subunit protein non-heme iron, heme b & heme c1 proton pump 4H+/2e-1 [see note 2]
cytochrome c
(ferrous / ferric)
inter-membrane space heme c mobile carrier
cytochrome c oxidase (complex 4) membrane spanning multi-subunit protein copper, heme a
& heme a3
proton pump 2H+/2e-1 [see note 2]
F0 / F1 ATPase
(ATP synthetase)
membrane spanning multi-subunit protein none proton pump
3H+ / ATP

Note 1: In addition to succinate dehydrogenase, several other enzymes contribute electrons to directly to ubiquinone. Acyl CoA dehydrogenase is a soluble flavoprotein involved in fatty acid oxidation in the mitochondrial matrix space. It feeds its reducing equivalents to ubiquinone via an intermediate low molecular weight electron transferring flavoprotein (ETF). Glycerol phosphate "oxidase" is another flavoprotein which also feeds reducing equivalents to ubiquinone. It is an inner membrane protein, but the relevant active centre faces outwards and reacts with its substrate in the cytosolic compartment, not in the matrix space. None of these enzymes contributes to proton pumping, so the P:O ratio from their substrates is only1.5

Note 2: The proton pumping stoichiometry for complex 3 and complex 4 is difficult because the electron acceptor for complex 3 (cytochrome c) is located on the outside of the inner membrane, whereas the oxidant for complex 4 (molecular oxygen) is reduced on the inside face of the inner membrane. It is necessary to count the negative charges on the electrons traversing the chain as well as the positive charges on the protons. Moreover, reduction of oxygen consumes two additional "chemical" protons to form water, in addition to those pumped by complex 4. These "chemical" protons don't count because they exactly balance the two "chemical" protons produced earlier when the hydrogen atoms were first removed during substrate oxidation. The net effect of all this is that complex 3 exports four protons but only two positive charges for each pair of electrons traversing the respiratory chain, while complex 4 exports two protons but four positive charges.

Liposomes are membranous vesicles which form spontaneously when many phospholipids are dispersed in aqueous media. If the lipids are simply shaken with water large multi-layered structures are produced with an "onion skin" arrangement. If the multi-layered liposomes are treated with ultrasonic vibrations they yield much smaller unilamellar vesicles with a simple limiting membrane composed of a single phospholipid bilayer. Both single and multi-layered liposomes can be prepared which are impermeable to the majority of charged ions, although uncharged, hydrophobic molecules have ready access to the interior. A small volume of the original preparation medium becomes trapped within each liposome as the membrane seals up.

Reconstitution: If hydrophobic membrane proteins are included during the lipid sonication step, these are incorporated into the liposome membranes. This has been used to re-constitute the respiratory chain from purified fragments. Unfortunately the proteins adopt a random orientation as they are incorporated into the liposome membrane, so half of them finish facing the wrong way round. It is therefore difficult to re-create a proton gradient. A variety of ingenious techniques have been used to select the correctly oriented protein population, for example by trapping the electron donor within the liposome and supplying the acceptor only to the external face.

Liposomes are also being actively researched as a means of delivering cytotoxic drugs to tumour cells, which have been targeted with monoclonal antibodies.

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