Coupling of Ox/Phos Under Aerobic Conditions
A quick glance reveals that we have taken glucose a small fraction along the way of oxidizing every carbon in it to CO2 and H2O. The complete oxidation happens under aerobic conditions when the glycolytic pathway is followed by the Krebs cycle. Pyruvate formed in glycolysis enters the mitochondrial matrix, and gets oxidatively decarboxylated to a 2C molecule, acetyl-CoA by the enzyme pyruvate dehydrogenase.
Acetyl CoA then enters the Krebs cycle, also called the tricarboxylic acid (TCA) cycle. It is shown below.
The main oxidizing agent used during aerobic metabolism is NAD+ (although FAD is used in one step) which gets converted to NADH. Unless the NAD+ can be regenerated, glycolysis and the Krebs cycle will grind to a halt. Luckily, under these conditions we are actually continually breathing one of the best oxidizing agents around, dioxygen. NADH is oxidized back to NAD+ not directly by dioxygen, but indirectly as electrons flow from NADH through a series of electron carriers to dioxygen, which gets reduced to water. This process is called electron transport. No atoms of oxygen are incorporated into NADH or any intermediary electron carrier. Hence the enzyme involved in the terminal electron transport step, in which electrons pass to dioxygen, is an oxidase. The enzymes of the Krebs cycle and electron transport are localized in mitochondria.
By analogy to the coupling mechanism under anaerobic conditions, it would be useful from a biological perspective if this electron transport from NADH to dioxygen, a thermodynamically favorable reaction (as you calculated in the last study guide - a value of about -55 kcal/mol), were coupled to ATP synthesis. It is! For years scientists tried to find a high energy phosphorylated intermediate, similar to that formed by glyceraldehyde-3-phosphate dehydrogenase in glycolysis, which could drive ATP synthesis (which likewise occurs in the mitochondria). None could be found. A startling hypothesis was put forward by Peter Mitchell, which was proven correct and for which he was awarded the Nobel Prize in Chemistry in 1978. The immediate source of energy to drive ATP synthesis was shown to come not from a phosphorylated intermediate, but a proton gradient across the mitochondrial inner membrane. All the enzyme complexes in electron transport are in the inner membrane of the mitochondria, as opposed to the cytoplasmic enzymes of glycolysis. A pH gradient is formed across the inner membrane in respiring mitochondria. In electron transport, electrons are passed from mobile electron carriers through membrane complexes back to another mobile carrier. Initially, NADH shuttles electrons (2 electron oxidation, characteristic of NAD+/NADH), to a flavin derivative, FMN, covalently attached to Complex I. The reduced form of FMN then passes electrons in single electron steps (characteristic of FAD-like molecules, which can undergo 1 or 2 electrons transfers) through the complex to the lipophilic electron carrier, ubiquinone (UQ).
This then passes electrons through Complex III to another mobile electron carrier, a small protein, cytochrome C. Then cytochrome C passes electrons through complex IV, cytochrome C oxidase, to dioxygen to form water. At each step electrons are passed to better and better oxidizing agents, as reflected in their increasing positive standard reduction potential. Hence the oxidation at each complex is thermodynamically favored.
- Jmol: Cytochrome C Oxidase
Complex II (also called succinate:quinone oxidoreductase) is a Krebs cycle enzyme that catalyzes the oxidation of succinate to fumarate by bound FAD (hence its other name: succinate dehydrogenase). It is not involved in the flow of electrons from NADH to dioxygen described above but passes electrons from the reduced succinate to ubiquinone to form fumarate and reduced ubiquinone which then can transfer electrons to cytochrome C through Complex III. The crystal structure of this complex has recently been solved by Yankovskaya et al. who have shown that the arrangement of the redox-active sites in the complex minimizes potential oxidation of bound FADH2 by dioxygen, minimizing production of harmful reactive oxygen species like superoxide.
At each complex, the energy released by the oxidative event is used to drive protons through each complex from the matrix to the intermembrane space of the mitochondria, and is not used to form a high energy mixed anhydride as we saw in the glyceraldehyde-3-phosphate dehydrogenase reaction. The actual mechanism of proton transfer is unclear.
How is this proton gradient coupled to ATP synthesis? Another mitochondrial inner membrane complex, FoF1ATPase, also called ATP synthase or complex V, is found in the inner mitochondrial membrane.
It contains two domains, a transmembrane proton channel, and an enzymatic domain which can either synthesize or hydrolyze ATP. As protons stream through the membrane pore, conformational changes, probably mediated by concerted changes in amino acid side chain pKa's, cause the protein to synthesize ATP. Based on kinetic and structural data, Boyer devised an innovative hypothesis for the mechanism of ATP synthesis, which has been supported by recent structural data. In this model the enzyme, which has multiple subunits, has 3 sites for ATP binding, named L, O, and T. The L or Loose site binds ATP loosely, the T or Tight site binds it tightly, while the O or Open site does not bind ATP. Although the ΔGo for ATP synthesis in solution is +7.5 kcal/mol, it appears the ΔGo for bound ADP + Pi ----> bound ATP is about 1. Hence the reaction is readily reversible. The difficulty lies in dissociating the bound ATP from the complex. Initially, ADP and Pi bind to the L site. A conformational change occurs, switching the site from L to T, and concomitantly, a T site with ATP bound to an O site which promotes ATP departure. Since the T site has ADP and Pi bound, but has high affinity for ATP, it promotes the synthesis of ATP at that site. This reflects the idea that enzymes bind the transition state (which presumably looks more like ATP than ADP and Pi) more tightly than the substrate. ADP and Pi bind to the newly formed L site, which promotes the switch from the T to O site, releasing ATP from the enzyme.
It should now be clear why the enzymes for oxidative phosphorylation in aerobic conditions are membrane bound. Only in this way could a proton gradient be established. Protons must be vectorially transferred in only one direction for a gradient to be established!
Aerobic ATP production can be uncoupled from electron transfer, as we saw with arsenate uncoupling of ox/phos in anaerobic metabolism in glycolysis. In that case, the energy source driving ATP synthesis was removed through hydrolysis of the mixed carboxylate/arsenate anhydride. In aerobic metabolism, the energy source is the proton gradient. If this gradient could be artificially collapsed, ATP synthesis would stop, but electron transport (oxidation of NADH through formation of water from dioxygen) would continue. 2,4-dinitrophenol can collapse the proton gradient and act as an uncoupler. In the low pH milieu of the intermembrane space, this weak acid would be protonated. It is also sufficiently nonpolar so as to have reasonable bilayer permeability. When it reaches the higher pH matrix, it can deprotonate. The net effect is to shunt protons through the intermembrane and not through the F0F1ATPase.
Figure: Uncoupling Aerobic Ox/Phos