The first enzyme in the electron transport chain is the NADH-CoQ oxidoreductase, also known as NADH dehydrogenase or complex I, which is the first entry of protons through the electron transport chain. As two electrons pass through complex I, four protons are pumped from the mitochondrial matrix into the intermembrane space. The second enzyme that allows protons to passes through the electron transport chain is succinic-coenzyme Q oxidoreductase, also known as succinate dehydrogenase or complex II.
It catalyzes the oxidation of succinic acid to form fumarate and the reduction of coenzyme Q10 to ubiquinone QH2. This reaction does not involve the transfer of electrons, nor does it pump out protons, providing less energy to compare with the oxidation process of NADH. The third entry to the proton on the electron transport chain is electron transfer flavin-coenzyme Q oxidoreductase, also known as electron transfer flavin dehydrogenase, which reduces Q10 by using electrons from electron transfer flavin in the mitochondrial matrix.
Coenzyme Q-cytochrome C reductase, also known as complex III, catalyzes the oxidation of QH2, and the reduction of cytochrome c and ferritin. In this reaction, cytochrome C carries an electron. Coenzyme Q is reduced to QH2 on one side of the mitochondrial membrane, while QH2 is oxidized to coenzyme Q10 on the other side, resulting in the transfer of protons on the membrane, which also contributes to the formation of proton gradients.
The last protein complex in the electron transport chain is cytochrome c oxidase, also called complex IV. It mediates the final reaction on the electron transport chain - transferring electrons to the final electron receptor oxygen - oxygen reduces to water - pumping protons through the membrane.
At the end of this reaction, protons that directly pumped out and that consumed by the reduction of oxygen to water increase the proton gradient. There is another electron-donating molecule - FADH2 in eukaryotes. FADH2 is also the intermediate metabolite during the earlier stage of cellular respiration such as glycolysis or citric acid cycle. And this reaction does not pump out protons either.
The subsequent reactions are nearly the same as those in the NADH2 electron transport chain. Prokaryotes such as bacteria and archaea have many electron transfer enzymes that can use a very wide range of chemicals as substrates.
As the same with eukaryotes, electron transport in prokaryotic cells also uses the energy released by oxidation from the substrate to pump protons across the membrane to create an electrochemical gradient, which drives ATP synthase to generate ATP. The difference is that bacteria and archaea use many different substrates as electron donors or electron receptors.
This also helps prokaryotes to survive and grow in different environments. Under normal conditions, electron transfer and phosphorylation are tightly coupled. Some compounds can affect electron transport or interfere with phosphorylation reactions, all of which cause oxidative phosphorylation abnormalities.
Here introduce four factors affecting oxidative phosphorylation. Respiratory chain inhibitor: A substance that blocks electron transport at a certain part of the respiratory chain and inhibits the oxidation process. Some substances inhibit the electron transfer between Cytb and Cytc1 , such as antimycin A and dimercaptopropanol. Cyanide, azide, H2S, and C0 inhibit cytochrome oxidase, making electrons unable to pass to oxygen. Living systems couple reactions in several ways, but the most common method of coupling reactions is to carry out both reactions on the same enzyme.
Consider again the phosphorylation of glycerol Equations Glycerol is phosphorylated by the enzyme glycerol kinase, which is found in your liver.
The product of glycerol phosporylation, glycerolphosphate Equation 2 , is used in the synthesis of phospholipids. Glycerol kinase is a large protein comprised of about amino acids. X-ray crystallography of the protein shows us that there is a deep groove or cleft in the protein where glycerol and ATP attach see Figure 6, below. Because the enzyme holds the ATP and the glycerol in place, the phosphate can be transferred directly from the ATP to glycerol.
Instead of two separate reactions where ATP loses a phosphate Equation 3 and glycerol picks up a phosphate Equation 2 , the enzyme allows the phosphate to move directly from ATP to glycerol Equation 4.
The coupling in oxidative phosphorylation uses a more complicated and amazing! This is a schematic representation of ATP and glycerol bound attached to glycerol kinase. The enzyme glycerol kinase is a dimer consists of two identical subuits. There is a deep cleft between the subunits where ATP and glycerol bind. Since the ATP and phosphate are physically so close together when they are bound to the enzyme, the phosphate can be transferred directly from ATP to glycerol. Hence, the processes of ATP losing a phosphate spontaneous and glycerol gaining a phosphate nonspontaneous are linked together as one spontaneous process.
Neglecting any differences in difficulty synthesizing or accessing these molecules by biological systems, rank the molecules in order of their efficiency as a free-energy currency i.
In order to couple the redox and phosphorylation reactions needed for ATP synthesis in the body, there must be some mechanism linking the reactions together. In cells, this is accomplished through an elegant proton-pumping system that occurs inside special double-membrane-bound organelles specialized cellular components known as mitochondria.
A number of proteins are required to maintain this proton-pumping system and catalyze the oxidative and phosphorylation reactions. There are three key steps in this process:.
Note: Steps a and b show cytochrome oxidase, the final electron-carrier protein in the electron-transport chain described above. When this protein accepts an electron green from another protein in the electron-transport chain, an Fe III ion in the center of a heme group purple embedded in the protein is reduced to Fe II. Cells use a proton-pumping system made up of proteins inside the mitochondria to generate ATP.
Before we examine the details of ATP synthesis, we shall step back and look at the big picture by exploring the structure and function of the mitochondria, where oxidative phosphorylation occurs. The mitochondria Figure 8 are where the oxidative-phosphorylation reactions occur. Mitochondria are present in virtually every cell of the body.
They contain the enzymes required for the citric-acid cycle the last steps in the breakdown of glucose , oxidative phosphorylation, and the oxidation of fatty acids. This is a schematic diagram showing the membranes of the mitochondrion. The purple shapes on the inner membrane represent proteins, which are described in the section below.
An enlargement of the boxed portion of the inner membrane in this figure is shown in Figure 8, below. The mitochondrial membranes are crucial for this organelle's role in oxidative phosphorylation. As shown in Figure 8, mitochondria have two membranes, an inner and an outer membrane. The outer membrane is permeable to most small molecules and ions, because it contains large protein channels called porins. The inner membrane is impermeable to most ions and polar molecules.
The inner membrane is the site of oxidative phosphorylation. Recall the discussion of protein channels in the " Maintaining the Body's Chemistry: Dialysis in the Kidneys " Tutorial. As shown in Figure 8, inside the inner membrane is a space known as the matrix ; the space between the two membranes is known as the intermembrane space.
This charge difference is used to provide free energy G for the phosphorylation reaction Equation 8. Electrons are not transferred directly from NADH to O 2 , but rather pass through a series of intermediate electron carriers in the inner membrane of the mitochondrion. This allows something very important to occur: the pumping of protons across the inner membrane of the mitochondrion.
As we shall see, it is this proton pumping that is ultimately responsible for coupling the oxidation-reduction reaction to ATP synthesis. Two major types of mitochondrial proteins see Figure 9, below are required for oxidative phosphorylation to occur. Both classes of proteins are located in the inner mitochondrial membrane.
The electron carriers can be divided into three protein complexes NADH-Q reductase 1 , cytochrome reductase 3 , and cytochrome oxidase 5 that pump protons from the matrix to the intermembrane space, and two mobile carriers ubiquinone 2 and cytochrome c 4 that transfer electrons between the three proton-pumping complexes.
Gold numbers refer to the labels on each protein in Figure 9, below. Because electrons move from one carrier to another until they are finally transferred to O 2 , the electron carriers shown in Figure 9,below are said to form an electron-transport chain.
Figure 9, below, is a schematic representation of the proteins involved in oxidative phosphorylation. To see an animation of oxidative phosphorylation, click on "View the Movie. This is a schematic diagram illustrating the transfer of electrons from NADH, through the electron carriers in the electron transport chain, to molecular oxygen.
Please click on the pink button below to view a QuickTime animation of the functions of the proteins embedded in the inner mitochondrial membrane that are necessary for oxidative phosphorylation.
Click the blue button below to download QuickTime 4. Ubiquinone Q 2 and cytochrome c Cyt C 4 are mobile electron carriers. Ubiquinone is not actually a protein. All of the electron carriers are shown in purple, with lighter shades representing increasingly higher reduction potentials.
The path of the electrons is shown with the green dotted line. ATP synthetase red has two components: a proton channel allowing diffusion of protons down a concentration gradient, from the intermembrane space to the matrix , and a catalytic component to catalyze the formation of ATP. For a more complete description of each step in oxidative phosphorylation indicated by the gold numbers , click here.
Click here for a brief description of each of the electron carriers in the electron-transport chain. It is important to note that, although NADH donates two electrons and O 2 ultimately accepts four electrons, each of the carriers can only transfer one electron at a time.
Hence, there are several points along the chain where electrons can be collected and dispersed. For the sake of simplicity, these points are not described in this tutorial. In the section above, we see that the oxidation-reduction process is a series of electron transfers that occurs spontaneously and produces a proton gradient. Why are the electron tranfers from one electron carrier to the next spontaneous? What causes electrons to be transferred down the electron-transport chain?
As seen in Table 2, below, and Figure 7a, in these carriers, the species being oxidized or reduced is Fe, which is found either in a iron-sulfur Fe-S group or in a heme group. Table 2 shows that the electrons are transferred through the electron-transport chain because of the difference in the reduction potential of the electron carriers. As explained in the green box below, the higher the electrical potential e of a reduction half reaction is, the greater the tendency is for the species to accept an electron.
Hence, in the electron-transport chain, electrons are transferred spontaneously from carriers whose reduction results in a small electrical potential change to carriers whose reduction results in an increasingly larger electrical potential change. An oxidation-reduction reaction consists of an oxidation half reaction and a reduction half reaction. Every half reaction has an electrical potential e. By convention, all half reactions are written as reductions, and the electrical potential for an oxidation half-reaction is equal in magnitude, but opposite in sign, to the electrical potential for the corresponding reduction i.
The electrical potential for an oxidation-reduction reaction is calculated by. Healthy host cells express MHC class I molecules but low levels of activating ligands, thus delivering overall stronger inhibitory signals to prevent NK cells from being activated.
However, unhealthy cells, including tumor and virus-infected cells, upregulate stress-induced activating ligands, or down-regulate their MHC class I molecules to escape from cytotoxic T cell killing. Hence, the activating signals override the inhibitory signals received by NK cells, leading to NK cell activation and the elimination of unhealthy cells 8.
Activated immune cells have high demands for ATP molecules for energy consumption and nutrients for anabolic synthesis to cater for their effector functions Glycolysis converts glucose into pyruvate via a series of metabolic reactions.
However, glycolysis is found to be the dominant metabolic pathway in pro-inflammatory cells, possibly because it could be rapidly activated via the induction of glycolytic enzymes and could provide intermediates for cell biosynthesis Briefly, blood samples were obtained from healthy donors with written consent and were approved by the Institutional Review Board of National University of Singapore At day 7, NK cells were re-stimulated by K feeder cells at the ratio of At day 14, NK cells were selectively expanded to about 1,fold and were used for experiments.
Anti-2B4 clone C1. After incubation, plates were washed once with PBS. Cells were harvested for subsequent metabolic and flow cytometry analyses.
ECAR was measured under basal conditions followed by sequential addition of 10 mM glucose, 0. This procedure allows an estimation of extracellular acidification caused by non-glycolytic acidification, glycolysis, and glycolytic capacity of NK cells.
This protocol allows the accurate calculation of oxygen consumption due to basal respiration, maximal respiration, ATP production and non-mitochondrial respiration. Cells were treated with 2-DG 30 mM , or oligomycin 2. When indicated, the pretreated NK cells were washed twice with PBS before stimulated with K cells at effector to target E:T ratio of for 30 min.
Cells were then harvested and stained for 20 min on ice with saturating concentration of antibodies for surface staining. Cells were then collected and washed with ice-cold PBS once. GAPDH housekeeping gene was used as the internal standard. The following primers were used:. A melting curve was performed at the end to confirm the specificity of the amplification. Each reaction was analyzed in duplicates.
NK cells were pretreated with 2-DG 30 mM , or oligomycin 2. Cells were then co-cultured with cell tracer violet-labeled K cells at E:T ratio of 0. When indicated, pretreated NK cells were washed with PBS twice before co-culturing with K cells in medium without metabolic inhibitors. For Kpretreated killing assay, K cells were treated with metabolic inhibitors stated above for 1 h and washed twice with PBS before co-culturing with untreated NK cells for 1 h at E:T ratio of 0. To detect dead cells, cells in the co-culture were collected and labeled with fixable viability dye eFluor FVD, eBioscience in PBS for 10 min at room temperature before flow cytometry analysis.
The percentage of killing by NK cells was calculated using the following equation:. Statistical analysis was performed with GraphPad Prism 6. Independent sample Student's t -test was used to compare the means of 2 groups. One-way and two-way ANOVA tests were used to compare means for more than 2 groups, and Dunnett's tests were used for multiple comparisons. Ex vivo expanded NK cells are widely used in NK-cell based immunotherapy in autologous and allogeneic transfer settings 8.
This expansion process is achieved by co-culturing NK cells with engineered feeder cells that express membrane-bound mb cytokines and co-stimulatory molecules. After expansion, NK cells are enriched in number and are more potent in eliminating tumor cells Figure 1. Glycolysis was quantified as the difference of non-glycolytic acidification and ECAR after addition of glucose; glycolytic capacity is calculated by the change of ECAR before and after addition of 2-DG E.
Basal respiration was quantified as the difference of non-mitochondrial respiration and OCR before addition of oligo. The unstimulated cells without 2-NBDG incubation denoted unlabeled served as the negative control. Glycolytic capacity represents the maximum rate of glycolysis that cells can achieve. The ATP-linked respiration was low in unstimulated ex vivo expanded NK cells, which further indicated that these cells relied heavily on glycolysis for ATP production.
The maximal respiration can be assessed by the addition of an uncoupler FCCP, which drives the respiratory chain to operate at maximum capacity. Because the expansion of NK cells using feeder cells would probably change the metabolic state of NK cells, thus the expanded NK cells may exhibit different metabolic responses from resting primary human NK cells upon NKR simulation.
Figure 2. E Quantification of glycolysis and glycolytic capacity of unstimulated and stimulated NK cells. One of the major effector functions of NK cells is the secretion of cytokines, e. We treated NK cells with 2-deoxyglucose 2-DG , which is an analog of glucose that blocks glycolysis. Figure 3.
As cytotoxicity is another important effector function of NK cell, we proceeded to explore how cell metabolism affects NK cell cytotoxicity. Due to limiting access to freshly isolated primary human NK cells, we mainly focused on ex vivo expanded NK cells in assessing NK cell cytotoxicity.
This approach is also consistent with the protocol adapted for cell therapy, i. We first analyzed the effect of glycolysis inhibition on NK cell cytotoxicity. We started with the short-term inhibition of glycolysis by treating NK cells with 2-DG for 4 h. We next introduced a longer-term inhibition of glycolysis by incubating NK cells with glucose-free medium overnight.
Inhibiting glycolysis by glucose starvation significantly reduced the killing of K cells by NK cells at all E:T ratios examined, with the most drastic decrease observed at E:T ratio of Figures 4A,D.
These data indicate that long-term inhibition of glycolysis impaired NK cell cytotoxicity. Figure 4.
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