 Pathway Diagrams |
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| Chem 431 |
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Fall 2001 |
| Lecture Notes:: 28 November |
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| PREVIOUS |
With the electron transport components we looked at last time a path for the transfer of electrons from substrate to oxygen has been created,
but we want more than a wire: want to use this flow of electrons to run a couple of "motors" to pump protons across the inner mitohondrial membrane, forming an electrochemical gradient, as shown in Figure 17.7, p 499. [overhead 18.8, lower] Note that on this diagram the heights of the boxes indicates the change in standard potential or energy. From this we can see that complexes I and IV obviously have enough energy change to support the phosphorylation of ADP to ATP, while complex III is marginal and complex II obviously does not have sufficient energy change.
But how is this energy captured? Mitochondria appear to be using the energy of moving the electrons through this potential gradient to pump protons across the inner mitochondrial membrane. (Figure 17.8 p 501) [overhead 18.7] The resulting proton motive gradient can then be used to make ATP as we shall see later. Lets look at the four complexes and what seems to be occurring in each.
Complex I: Note that the electrons are received as a pair (a hydride ion) on NADH, but most carriers can only handle single electrons so FMN acts as a transducer, picking up a pair of electrons, but passing them on singly to the FeS centers in this complex. The electrons are then passed singly on to CoQ, which, like FMN can carry either pairs or single electrons. Four H+ are pumped across the inner mitochondrial membrane by complex I. (Note that formally the protons from NADH and H+ can be considered as going to FMNH2 and the UQH2.)
Order of components of ETS {overhead 20-10, V&V, 21.30 G&G}.
The order shown has been discovered via wide variety of studies. Conveniently it turns out that the components are ordered as one would expect from their standard reduction potentials. (A state of affairs which is certainly not necessary, since it is the actual, not the standard, reduction potential which matters in the flow of electrons through this system.) The relative reduction potentials of the four complexes is illustrated in Figure 17.7 on p 499. [overhead 18.8, lower] Can use specific inhibitors of complexes and cross-over experiments to place components in groups before and after the inhibition. With a number of inhibitors the order can then be logically determined. Finally, with restricted [ADP] will see variation with relative redox state, with components grouped by order of ATP/ETS coupling (proton pumping locations).
ATP Synthase (Complex V; F0F1 ATP synthase, Figure 17.19, p 514) [overhead 21.25 G&G]: ATP synthase uses the proton gradient to make ATP from ADP and Pi. It is bound to the inner membrane and has a characteristic knob and stalk structure as seen in electron micrographs. It can be broken into two multi protein components: The F1 component (the "knob") hydrolyses ATP when it is isolated by itself and is referred to as F1 ATPase. The F0 component is a membrane spanning proton channel. When the two components are linked the passage of protons through the channel is coupled to ATP synthesis. According to the binding-change mechanism there are three sites in the alpha3beta3 oligomer of the knob. At any given time the three sites are in three different conformations, as shown in Figure 17.21, p 516: open, loose, or tight. [overhead 18-18, P] Each site passes sequentially through the three conformations, apparently while physically rotating 120° for each change. Following one site: 1) ADP and Pi bind to the site in the open conformation. 2) Passage of 3 protons through the channel causes the ab oligomer to rotate 120° and change to the loose or L conformation, , holding the ADP and Pi (all three active sites to go to the next conformation simultaneously). 3) Passage of another 3 protons through the channel causes the ab oligomer to rotate 120° and change to the tight conformation with consequent condensation of ADP and Pi to ATP. 4) Passage of another 3 protons through the channel causes the ab oligomer to rotate 120° and change to the open conformation, releasing ATP. Note the net result of 3 protons/ATP.
(For current view and evidence on ETS and OxPhos see M. Saraste Science 283 (5 March 1999) pp 1488-93; for ATPase 'motor' see Paul D. Boyer (18 Nov 1999) "What matkes ATP synthase spin?" Nature 402: 247-8.)
Given the requirement for a very tight inner-mitochondrial membrane in order to maintain the proton electrochemical gradient, how do important charged molecules such as ATP and NADH get across the membrane?
ADP & ATP are obviously among the most important substances to transport into and out of the mitochondria. Adenine nucleotide translocase exchanges matrix ATP for cytosolic ADP in their magnesium-free forms. Note that in this exchange ATP4- leaves the matrix as ADP3- goes in, resulting in a net loss of (1-) for the matrix. This increases the charge gradient across the membrane, and thus must be driven by the mitochondrial proton gradient. Of course Pi must also be transported across the membrane with ADP to make ATP in the matrix. This is accomplished using another transporter which co-transports a dihydrogen phosphate and a single proton in an electroneutral process. Note the addition of these two processes is equivalent to moving one proton from the cytosol to the matrix, costing the gradient one proton (moving a negative charge out is equivalent to moving a positive charge out).
The net cost of providing an ATP to the cytosol is thus four protons: three to convert ADP + Pi to ATP and one to transport ATP out of, while bringing ADP and Pi into, the matrix. (Note that this means that bacteria may get more ATP (up to 38 ATP's instead of the32 expected in mammals).
| Pathway Diagrams |
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