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During anaerobic respiration, why are electrons carried by NADH not transferred to the electron transport chain (ETC)? What happens is that lactate dehydrogenase reduces pyruvate to lactate, while removing H+ from NADH to form NAD+.
Why doesn't pyruvate simply take the place of O2 as the final electron acceptor in the ETC during oxidative phosphorylation? Electrons would still be able to flow through the ETC and allow for the regeneration of NAD+, wouldn't they?
Someone else may come along later with a definitive answer but I found this question intriguing so here are my thoughts:
The standard redox potentials of the mitochondrial ETC carriers are:
NAD⁺/NADH -0.32 V complex I (Fe-S) -0.27 V complex II (cyt b₅₆₀) -0.08 V complex III ((cyt c₁) +0.23 V complex IV (cyt a₃) +0.38 V O₂/H₂O +0.82 V
Note that electron transport is carried out from negative to positive.
Now, for pyruvate/lactate the standard redox potential is -0.19 V so on the basis of this pyruvate could in theory accept electrons from complex I, but not any further down the chain. However, complex I normally transfers electrons to coenzyme Q within the membrane. The standard redox potential of CoQ is +0.04 V which is already too positive to be able to then reduce pyruvate at the membrane surface. Thus there would have to be a novel way of transferring electrons from the final FeS centre of complex I, which is within the membrane, to pyruvate. Pyruvate is, of course, soluble and is generated by cytoplasmic glycolysis, so this transfer would have be at the membrane surface that faces the intermembrane space of the mitochondria.
If such a scheme had evolved then it might be possible to achieve some proton pumping through the novel version of complex I, which would be energetically advantageous.
Another complication is - assuming that a complex I with pyruvate as electron acceptor had evolved - how would the cell/ mitochondrion regulate electron flow during aerobic respiration? Presumably the pyruvate-accepting version of complex I would have to be kept inactive somehow until needed.
Clearly it is much simpler to have a soluble lactate dehydrogenase to deal with the pyruvate and regenerate NAD⁺.
In anaerobic respiration the free energy change of the reoxidation of NADH by pyruvate is less than would be required to phosphorylate a molecule of ADP to ATP. The purpose of the electron transport chain is to harness the much greater free energy change in the oxidation of NADH by molecular oxygen. So pyruvate can neither “take the place of O2 as the final electron acceptor”, nor would there be any point in modifying this complex machinery just so that it could be used by pyruvate to regenerate NAD+ when a single cytoplasmic enzyme (lactate dehydrogenase) will do the job.
More detailed numerical answer
This answer is taken from section 18.2 of Berg et al. and involves calculations of free energy changes from the redox potentials of the different half reactions and their relationship to the free energy of hydrolysis of ATP. It is worth careful reading, but I will summarize the key points.
- Both pyruvate and oxygen can oxidize NADH, but the concomitant Standard Gibbs Free Energy change is very different in the two cases. This is the crux of the matter.
- The standard redox potential for NADH → NAD+ is +0.32 V
- The standard redox potential for Pyruvate → Lactate is -0.19 V
- The standard redox potential for O2 → H2O is +0.82
Combining these half-reaction redox potentials in the two oxidation reactions and then converting to standard free energy change (ΔG˚ʹ):
- Oxidation of NADH by Pyruvate: ΔG˚ʹ = -6.0 kcal/mol
- Oxidation of NADH by Oxygen: ΔG˚ʹ = -52.6 kcal/mol
But ΔG˚ʹ for ADP → ATP = -7.5 kcal/mol
So it can be seen that the energetics of the oxidation of NADH by pyruvate do not yield enough energy to synthesize even a molecule of ATP, never mind the approx. 3 that are obtained from oxygen. The electron transport chain is a device for breaking up this latter oxidation reaction into stages so that the free energy change can be used for generating the proton gradient that is used to generate ATP. It can only work with a powerful enough oxidizing agent.
The oxidation of NADH by pyruvate is useful, but only to regenerate NAD+ to allow glycolysis to continue and generate a smaller amount of ATP by substrate-level phosphorylation made possible by the glyceraldehyde 3-phosphate dehydrogenase reaction. For this oxidation only a simple enzyme, lactate dehydrogenase is required. There is certainly no reason to transport the pyruvate into the mitochondria (assuming they exist - think erythrocytes), which process, though incompletely understood, may well have an energetic cost.
During the process, a proton gradient is created when the protons are pumped from the mitochondrial matrix into the intermembrane space of the cell, which also helps in driving ATP production. Often, the use of a proton gradient is referred to as the chemiosmotic mechanism that drives ATP synthesis since it relies on a higher concentration of protons to generate “proton motive force”. The amount of ATP created is directly proportional to the number of protons that are pumped across the inner mitochondrial membrane.
The electron transport chain involves a series of redox reactions that relies on protein complexes to transfer electrons from a donor molecule to an acceptor molecule. As a result of these reactions, the proton gradient is produced, enabling mechanical work to be converted into chemical energy, allowing ATP synthesis. The complexes are embedded in the inner mitochondrial membrane called the cristae in eukaryotes. Enclosed by the inner mitochondrial membrane is the matrix, which is where necessary enzymes such as pyruvate dehydrogenase and pyruvate carboxylase are located. The process can also be found in photosynthetic eukaryotes in the thylakoid membrane of chloroplasts and in prokaryotes, but with modifications.
By-products from other cycles and processes, like the citric acid cycle, amino acid oxidation, and fatty acid oxidation, are used in the electron transport chain. As seen in the overall redox reaction,
energy is released in an exothermic reaction when electrons are passed through the complexes three molecules of ATP are created. Phosphate located in the matrix is imported via the proton gradient, which is used to create more ATP. The process of generating more ATP via the phosphorylation of ADP is referred to oxidative phosphorylation since the energy of hydrogen oxygenation is used throughout the electron transport chain. The ATP generated from this reaction go on to power most cellular reactions necessary for life.
Nitrate Reduction and Denitrification
Denitrification is a type of anaerobic respiration that uses nitrate as an electron acceptor.
Outline the processes of nitrate reduction and denitrification and the organisms that utilize it
- Denitrification generally proceeds through a stepwise reduction of some combination of the following intermediate forms: NO3 − → NO2 − → NO + N2O → N2.
- Generally, several species of bacteria are involved in the complete reduction of nitrate to molecular nitrogen, and more than one enzymatic pathway has been identified in the reduction process.
- Complete denitrification is an environmentally significant process as some intermediates of denitrification (nitric oxide and nitrous oxide) are significant greenhouse gases that react with sunlight and ozone to produce nitric acid, a component of acid rain.
- electron acceptor: An electron acceptor is a chemical entity that accepts electrons transferred to it from another compound. It is an oxidizing agent that, by virtue of its accepting electrons, is itself reduced in the process.
- eutrophication: The process of becoming eutrophic.
- facultative: Not obligate optional, discretionary or elective
In anaerobic respiration, denitrification utilizes nitrate (NO3 – ) as a terminal electron acceptor in the respiratory electron transport chain. Denitrification is a widely used process many facultative anaerobes use denitrification because nitrate, like oxygen, has a high reduction potential
Denitrification is a microbially facilitated process involving the stepwise reduction of nitrate to nitrite (NO2 – ) nitric oxide (NO), nitrous oxide (N2O), and, eventually, to dinitrogen (N2) by the enzymes nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase. The complete denitrification process can be expressed as a redox reaction: 2 NO 3− + 10 e − + 12 H + → N2 + 6 H2O.
Protons are transported across the membrane by the initial NADH reductase, quinones and nitrous oxide reductase to produce the electrochemical gradient critical for respiration. Some organisms (e.g. E. coli) only produce nitrate reductase and therefore can accomplish only the first reduction leading to the accumulation of nitrite. Others (e.g. Paracoccus denitrificans or Pseudomonas stutzeri) reduce nitrate completely. Complete denitrification is an environmentally significant process because some intermediates of denitrification (nitric oxide and nitrous oxide) are significant greenhouse gases that react with sunlight and ozone to produce nitric acid, a component of acid rain. Denitrification is also important in biological wastewater treatment, where it can be used to reduce the amount of nitrogen released into the environment, thereby reducing eutrophication.
Denitrification takes place under special conditions in both terrestrial and marine ecosystems. In general, it occurs where oxygen is depleted and bacteria respire nitrate as a substitute terminal electron acceptor. Due to the high concentration of oxygen in our atmosphere, denitrification only takes place in anaerobic environments where oxygen consumption exceeds the oxygen supply and where sufficient quantities of nitrate are present. These environments may include certain soils and groundwater, wetlands, oil reservoirs, poorly ventilated corners of the ocean, and in sea floor sediments.
The role of soil bacteria in the Nitrogen cycle: Denitrification is an important process in maintaining ecosystems. Generally, denitrification takes place in environments depleted of oxygen.
Denitrification is performed primarily by heterotrophic bacteria (e.g. Paracoccus denitrificans), although autotrophic denitrifiers have also been identified (e.g., Thiobacillus denitrificans). Generally, several species of bacteria are involved in the complete reduction of nitrate to molecular nitrogen, and more than one enzymatic pathway have been identified in the reduction process.
Rhizobia are soil bacteria with the unique ability to establish a N2-fixing symbiosis on legume roots. When faced with a shortage of oxygen, some rhizobia species are able to switch from O2-respiration to using nitrates to support respiration.
The direct reduction of nitrate to ammonium (dissimilatory nitrate reduction) can be performed by organisms with the nrf- gene. This is a less common method of nitrate reduction than denitrification in most ecosystems. Other genes involved in denitrification include nir (nitrite reductase) and nos (nitrous oxide reductase), which are possessed by such organisms as Alcaligenes faecalis, Alcaligenes xylosoxidans, Pseudomonas spp, Bradyrhizobium japonicum, and Blastobacter denitrificans.
What is the final electron acceptor in the electron transport chain?
Read in-depth answer here. Likewise, what is the final electron acceptor in the electron transport chain quizlet?
(which relies on oxygen to act as the final electron acceptor in the electron transport chain. ADP is then phosphorylated to ATP by ATP synthase.)
Also, what molecule accepts electrons at the end of the electron transport chain? Oxygen
Similarly, you may ask, what is the final electron acceptor in cellular respiration?
Explanation: In cellular respiration, oxygen is the final electron acceptor. Oxygen accepts the electrons after they have passed through the electron transport chain and ATPase, the enzyme responsible for creating high-energy ATP molecules.
How many ATP are made in the electron transport chain?
Electron transport chain This stage produces most of the energy ( 34 ATP molecules, compared to only 2 ATP for glycolysis and 2 ATP for Krebs cycle). The electron transport chain takes place in the mitochondria. This stage converts the NADH into ATP.
In this class, most of the reduction/oxidation reactions (redox) that we discuss occur in metabolic pathways (connected sets of metabolic reactions). Here the cell breaks down the compounds it consumes into smaller parts and then reassembles them into larger macromolecules. Redox reactions also play critical roles in energy transfer, either from the environment or within the cell, in all known forms of life. For these reasons, it is important to develop at least an intuitive understanding and appreciation for redox reactions in biology.
Most students of biology will also study reduction and oxidation reactions in their chemistry courses these kinds of reactions are important well beyond biology. Regardless of the order in which students are introduced to this concept (chemistry first or biology first), most will find the topic presented in very different ways in chemistry and biology. That can be confusing.
Chemists often introduce the concepts of oxidation and reduction from the technically more correct and inclusive standpoint of oxidation states. See this link for more information: <https://chem.libretexts.org/Bookshel. ation_Numbers)>. Fortunately, there&rsquos no need to go into the details here (most of you will see that in chemistry at some point), just follow the argument for now. It might make things less confusing in both the long and short run. Anyhow, chemists will often ask students to apply a set of rules (see link above) to determine the oxidation states of individual atoms in a reaction. The chemistry formalism defines oxidation as an increase in oxidation state and reduction as a decrease in oxidation state.
All of this, of course, holds true in biology. However, biologists don&rsquot typically think of redox reactions in this way. Why? We suspect it&rsquos because most of the redox reactions encountered in biology involve a change in oxidation state that comes about because electrons are transferred between molecules. So, biologists typically define reduction as a gain of electrons and oxidation as a loss of electrons. The biological concept of redox is entirely consistent with the concept chemists use but it doesn&rsquot account for redox reactions that can happen without the transfer of electrons. The biologist&rsquos definition is therefore not as general as the chemist&rsquos, but it works for most cases encountered in biology.
This is a biology reading for a biology class. We, therefore, approach redox from the &ldquogain/loss of electrons&rdquo conceptualization that is commonly taught in biology classes. In our opinion, it&rsquos easier to use (no long list of rules to memorize and apply), more intuitive, and works for almost all cases we care about in undergraduate biology. So, if you had chemistry already and this topic seems a little different in biology, remember that at its core it&rsquos the same thing you learned about before. Biologists just adapted what you learned in chemistry to make more intuitive sense in biology. If you haven&rsquot learned about redox yet don&rsquot worry. If you can understand what we&rsquore trying to do here when you cover this concept in chemistry class you&rsquoll be a few steps ahead. You&rsquoll just need to generalize your thinking a bit instead of seeing the topic for the first time.
Let's start with some generic reactions
Transferring electrons between two compounds results in one of these compounds loosing an electron and one compound gaining an electron. For example, look at the figure below. If we use the energy story rubric to look at the overall reaction, we can compare the before and after characteristics of the reactants and products. What happens to the matter (stuff) before and after the reaction? Compound A starts as neutral and becomes positively charged. Compound B starts as neutral and becomes negatively charged. Because electrons are negatively charged, we can explain this reaction with the movement of an electron from Compound A to B. That is consistent with the changes in charge. Compound A loses an electron (becoming positively charged), and we say that A has become oxidized. For biologists, oxidation is associated with the loss of electron(s). B gains the electron (becoming negatively charged), and we say that B has become reduced. Reduction is associated with the gain of electrons. We also know, since a reaction occurred (something happened), that energy must have been transferred and/or reorganized in this process and we'll consider this shortly.
Figure 1.Generic redox reaction with half-reactions
To reiterate: When an electron(s) is lost, or a molecule is oxidized, the electron(s) must then pass to another molecule. We say that the molecule gaining the electron becomes reduced. Together these paired electron gain-loss reactions are known as an oxidation-reduction reaction (also called a redox reaction).
This idea of paired half-reactions is critical to the biological concept of redox. Electrons don&rsquot drop out of the universe for &ldquofree&rdquo to reduce a molecule or jump off a molecule into the ether. Donated electrons MUST come from a donor molecule and be transferred to some other acceptor molecule. For example in the figure above the electron the reduces molecule B in half-reaction 2 must come from a donor - it just doesn't appear from nowhere! Likewise, the electron that leaves A in half-reaction 1 above just "land" on another molecule - it doesn't just disappear from the universe.
Therefore, oxidation and reduction reactions must ALWAYS be paired. We&rsquoll examine this idea in more detail below when we discuss the idea of &ldquohalf-reactions&rdquo.
A tip to help you remember: The mnemonic LEO says GER (Lose Electrons = Oxidation and Gain Electrons = Reduction) can help you remember the biological definitions of oxidation and reduction.
Figure 2. A figure for the mnemonic "LEO the lion says GER." LEO: Loss of Electrons = Oxidation. GER: Gain of Electrons = Reduction
Attribution: Kamali Sripathi
&bull The vocabulary of redox can be confusing: Students studying redox chemistry can often become confused by the vocabulary used to describe the reactions. Terms like oxidation/oxidant and reduction/reductant look and sound very similar but mean distinctly different things. An electron donor is also sometimes called a reductant because it is the compound that causes the reduction (gain of electrons) of another compound (the oxidant). In other words, the reductant is donating it&rsquos electrons to the oxidant which is gaining those electrons. Conversely, the electron acceptor is called the oxidant because it is the compound that is causing the oxidation (loss of electrons) of the other compound. Again, this simply means the oxidant is gaining electrons from the reductant who is donating those electrons. Confused yet?
Yet another way to think about definitions is to remember that describing a compound as reduced/oxidized is describing the state that the compound itself is in, whereas labeling a compound as a reductant/oxidant describes how the compound can act, to either reduce or oxidize another compound. Keep in mind that the term reductant is also synonymous with reducing agent and oxidant is also synonymous with oxidizing agent. The chemists who developed this vocabulary need to be brought up on charges of "willful thickheadedness" at science trial and then be forced to explain to the rest of us why they needed to be so deliberately obtuse.
The confusing language of redox: quick summary
1. A compound can be described as &ldquoreduced&rdquo - term used to describe the compound's state
2. A compound can be a &ldquoreductant&rdquo - term used to describe a compound's capability (it can reduce something else). The synonymous term "reducing agent" can be used to describe the same capability (the term "agent" refers to the thing that can "do something" - in this case reduce another molecule).
3. A compound can be an &ldquooxidant&rdquo - term used to describe a compound's capability (it can oxidize something else). The synonymous term "oxidizing agent" can be used to describe the same capability (the term "agent" refers to the thing that can "do something" - in this case oxidize another molecule).
4. A compound can &ldquobecome reduced&rdquo or "become oxidized"- term used to describe the transition to a new state
Since all of these terms are used in biology, in General Biology we expect you to become familiar with this terminology. Try to learn it and use it as soon as possible - we will use the terms frequently and will not have the time to define terms each time.
Knowledge Check Quiz
The Half Reaction
Here we introduce the concept of the half reaction. We can think each half reaction as a description of what happens to one of the two molecules (i.e. the donor and the acceptor) involved in a "full" redox reaction. A "full" redox reaction requires two half reactions. We illustrate this below. In the example below, half reaction #1 depicts the molecule AH becoming losing two electrons and a proton and in the process becoming A + . This reaction depicts the oxidation of AH. Half reaction #2 depicts the molecule B + gaining two electrons and a proton to become BH. This reaction depicts the reduction of B + . Each of these two half reactions is conceptual and can't happen on their own. The electrons lost in half reaction #1 MUST go somewhere, they can't just disappear. Likewise, the electrons gained in half reaction #2 must come from something. They too just can't appear out of nowhere.
One can imagine that there might be different molecules that can serve as potential acceptors (the place for the electrons to go) for the electrons lost in half reaction #1. Likewise, there might be many potential reduced molecules that can serve as the electron donors (the source of electrons) for half reaction #2. In the example below, we show what happens (the reaction) when molecule AH is the donor of electrons for molecule B + . When we put the donor and acceptor half reactions together, we get a "full" redox reaction that can actually happen. In the figure below we call that reaction "Reaction #1". When this happens we call the two half reactions coupled.
Figure 3. Generic redox reaction where compound AH is being oxidized by compound B + . Each half reaction represents a single species or compound to either lose or gain electrons (and a subsequent proton as shown in the figure above). In half reaction #1 AH loses a proton and 2 electrons: in the second half reaction, B + gains 2 electrons and a proton. In this example HA is oxidized to A + while B + is reduced to BH.
Using this idea, we can theoretically couple and think about any two half reactions, one half reaction serving as the electron donor for the other half reaction that accepts the donated electrons. For instance, using the example above, we could consider coupling the reduction of B + that happens in half reaction 2 with another half reaction describing the oxidation of the molecule NADH. In that case, the NADH would be the electron donor for B + . Likewise you could couple the oxidation of AH that happens in half reaction #1 with a half reaction describing the the reduction of hypothetical molecule Z + . You can mix-and-match half reactions together as you please provided one half is describing the oxidation of a compound (it's donating electrons) and the reduction of another compound (it's accepting the donated electrons).
A note on how we write full reactions versus half reactions: In the example above, when we write Reaction #1 as an equation, the 2 electrons and the H + that are explicitly described in the underlying half reactions, are not explicitly included in the text of the full reaction. In the reaction above you must infer that an exchange of electrons happens. This can be observed by trying to balance charges between each reactant and it's corresponding product. Reactant AH becomes product A + . In this case, you can infer that some movement of electrons must have taken place. To balance the charges on this compound (make the sum of charges on each side of the equation equal) you need to add 2 electrons to the right side of the equation, one to account for the "+" charge on A + and a second to go with the H + that was also lost. The other reactant B + is converted to BH. It must therefore gain 2 electrons to balance charges, one for B + and a second for the additional H + that was added. Together this information leads you to conclude that the most likely thing to have happened is that two electrons were exchanged between AH and B + .
This will also be the case for most redox reactions in biology. Fortunately, in most cases, either the context of the reaction, the presence of chemical groups often engaged in redox (e.g. metal ions), or the presence of commonly used electron carriers (e.g. NAD + /NADH, FAD + /FADH2, ferredoxin, etc.) will alert you that the reaction is of class "redox". You will be expected to learn to recognize some of these common molecules.
By convention, we quantitatively characterize redox reactions using an measure called reduction potentials.The reduction potential attempts to quantitatively describe the &ldquoability&rdquo of a compound or molecule to gain or lose electrons. The specific value of the reduction potential is determined experimentally, but for the purpose of this course we assume that the reader will accept that the values in provided tables are reasonably correct. We can anthropomorphize the reduction potential by saying that it is related to the strength with which a compound can &ldquoattract&rdquo or &ldquopull&rdquo or &ldquocapture&rdquo electrons. Not surprisingly this is is related to but not identical to electronegativity.
What is this intrinsic property to attract electrons?
Different compounds, based on their structure and atomic composition have intrinsic and distinct attractions for electrons. This quality leads each molecule to have its own standard reduction potential or E0&rsquo. The reduction potential is a relative quantity (relative to some &ldquostandard&rdquo reaction). If a test compound has a stronger "attraction" to electrons than the standard (if the two competed, the test compound would "take" electrons from the standard compound), we say that the test compound has a positive reduction potential. The magnitude of the difference in E0&rsquo between any two compounds (including the standard) is proportional to how much more or less the compounds "want" electrons. The relative strength of the reduction potential is measured and reported in units of Volts (V)(sometimes written as electron volts or eV) or milliVolts (mV). The reference compound in most redox towers is H2.
Possible NB Discussion Point
Rephrase for yourself: How do you describe or think about the difference between the concept of electronegativity and red/ox potential?
Redox student misconception alert: The standard redox potential for a compound reports how strongly a substance wants to hold onto an electron in comparison to hydrogen. Since both redox potential and electronegativity are both discussed as measurements for how strongly something "wants" an electron, they are sometimes conflated or confused for one another. However, they are not. While the electronegativity of atoms in a molecule may influence its redox potential, it is not the only factor that does. You don't need to worry about how this works. For now, try to keep them as different and distinct ideas in your mind. The physical relationship between these two concepts is well beyond the scope of this general biology class.
The Redox Tower
All kinds of compounds can take part in redox reactions. Scientists have developed a graphical tool, the redox tower, to tabulate redox half reactions based on their E0 ' values. This tool can help predict the direction of electron flow between potential electron donors and acceptors and how much free energy change might be expected from a specific reaction. By convention, all half reactions in the table are written in the direction of reduction for each compound listed.
In the biology context, the electron tower usually ranks a variety of common compounds (their half reactions) from most negative E0 ' (compounds that readily get rid of electrons), to the most positive E0 ' (compounds most likely to accept electrons). The tower below lists the number of electrons that are transferred in each reaction. For example, the reduction of NAD + to NADH involves two electrons, written in the table as 2e - .
What is the final electron acceptor of the electron transport chain?
The final acceptor of electrons in the electron transport chain is molecular OXYGEN. Electron transport chains are used for extracting energy via redox reactions from sunlight in photosynthesis or, such as in the case of the oxidation of sugars, cellular respiration.
Similarly, what is the end result of the electron transport chain? The end products of the electron transport chain are water and ATP. A number of intermediate compounds of the citric acid cycle can be diverted into the anabolism of other biochemical molecules, such as nonessential amino acids, sugars, and lipids.
Likewise, people ask, what is a final electron acceptor?
A final or terminal electron acceptor is a molecule that accepts electrons right at the end of a chain of electron transfer. In aerobic respiration, the terminal electron acceptor is oxygen, which combines with two protons and the gained electrons (from the electron transport chain) to form water.
What is the final electron acceptor in oxidative phosphorylation?
The electrons are then drawn to oxygen, which is the final electron acceptor. Its important to note that oxygen must be present for oxidative phosphorylation to occur. Water is formed as oxygen receives the electrons from protein complex 4, and combines with protons on the inside of the cell.
Team:TU Delft-Leiden/Project/Life science/EET/theory
In the wet lab, we integrated the Electron Transport pathway of Shewanella oneidensis into Escherichia coli . Here you can find information with respect to literature consulted regarding implementation of the Electron Transport pathway in E. coli.
To facilitate extracellular electron transport in E. coli we genetically introduced a heterologous electron transport pathway of the metal-reducing bacterium S. oneidensis. The electron transfer pathway of S. oneidensis is comprised of C-type cytochromes that shuttle electrons from the inside to the outside of the cell . As a result, this bacterium couples the oxidation of organic matter to the reduction of insoluble metals during anaerobic respiration. There are several proteins that define the route for the electrons and thus are the major components of the electron transfer pathway (see figure 1). Our key-player proteins are CymA, an inner membrane cytochrome, MtrA, which is a periplasmic decaheme cytochrome, MtrC, an outer membrane decaheme cytochrome and MtrB, an outer membrane β-barrel protein.
Figure 1. Major components of the S. oneidensis electron transfer pathway. Via a series of intermolecular electron transfer events, e.g. from menaquinol to CymA, from CymA to MtrA, and from MtrA via membrane pore MtrB to MtrC, the electrons are transferred to the extracellular space. The electrons are derived from lactate oxidation by the enzyme(s) lactate dehydrogenase, of which several forms exist. NapC is a native E. coli cytochrome with comparable functionality to CymA (adapted from Goldbeck et al., 2013).
Now we have our so called Mtr electron conduit, but it will not function unless the multiple post-translational modifications are correctly carried out. The cytochrome C maturation (Ccm) proteins help the conduit proteins to mature properly by providing them with heme, which is one of the requirements to carry and transfer electrons . The step-by-step assembly of the Mtr protein complex is described in more detail in Deterministic Model of EET Complex Assembly and Integration of Departments, subsection ‘Relevant details of the extracellular electron transport system.
Jensen et al.(2010) have described a genetic strategy by which E. coli was capable to move intracellular electrons, resulting from metabolic oxidation reactions, to an inorganic extracellular acceptor by reconstituting a portion of the extracellular electron transfer chain of S. oneidensis . However, bacteria expressing the Mtr electron conduit showed impaired cell growth. To improve extracellular electron transfer in E. coli, Goldbeck et al. used an E. coli host with a more tunable expression system by using a panel of constitutive promoters. Thereby they generated a library of strains that separately transcribe the mtr- and ccm operons. Interestingly, the strain with improved cell growth and fewer morphological changes generated the largest maximal current per cfu (colony forming unit), rather than the strain with more MtrC and MtrA present . In the Module Electron Transport we aimed to reproduce the results reported by Goldbeck et al. in a BioBrick compatible way. To our knowledge we are the first iGEM team that successfully BioBricked the Mtr pathway. On top of that, we have succeeded to BioBrick mtrCAB under control of a weakened T7 promoter with the lac operator (T7 lacO) and the ccm cluster under control of the pFAB640 promoter, a combination that was found to generate the largest maximal current .
A Biosensor based on the Shewanella oneidensis Electron Transport pathway
Microbial Fuel Cell (MFC) based systems like the S. oneidensis Electron Transfer pathway are already introduced to the field of biosensors . An arabinose inducible promoter system was used as proof of principle in the above mentioned case. These results clearly showed that the current production depends on the addition of arabinose in a linear fashion. Therefore we believe that the implementation of the Electron Transfer pathway in E. coli has potential to develop itself as a quantitative and inexpensive biosensor.
Improved parts lead to Extracellular Electron Transport
E. coli strains expressing the extracellular electron transfer complex display limited control of MtrCAB expression. In addition, these strains show impaired cell growth . Use of a weakened T7 lacO promoter upstream of the mtrCAB cluster was shown to optimize the MtrCAB expression and reduce morphological perturbations . Therefore we aimed to improve the MtrCAB BioBrick BBa_K1172401 of the Bielefeld 2013 team by adding the weakened T7 lacO promoter. While characterizing their BioBrick, we could not detect the coding sequence of mtrCAB by restriction analysis nor sequencing. Therefore we started from scratch to clone the mtrCAB genes under control of the weakened T7 lacO promoter. We confirmed the sequence, and were able to show extracellular electron transport using our own mtrCAB BioBrick BBa_K1316012 (see figure 2).
Figure 2: Plasmid carrying the BBa_K1316012 BioBrick. BBa_K1316012 encodes a weakened T7 lacO promoter and the coding sequences of mtrC, mtrA and mtrB, indicated with grey arrows.
Carbon Metabolism and Electron Transport
Contemplated in this section is a general description of carbon metabolism of E. coli centered on generation of extracellular electron transport. Furthermore, several intriguing challenges as well as possible consequences with respect to these challenges are indicated.
Shewanella oneidensis natively hosts extracellular electron transfer pathway(s)
Shewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to utilize insoluble substrates for respiration purposes is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis .
Respiration in model organism Escherichia coli
Generation of extracellular electron transport (EET) by implementing and expressing genes of S. oneidensis in model organism Escherichia coli is directly influenced by the native carbon metabolism and poses, amongst others, the following questions which carbon sources can be used as electron donors? Are there possibilities to promote growth of E.coli and make possible EET in one go? Effectively, necessary for growth are ATP, (carbon) sources of elements essential to growth and proliferation as well as compounds that serve as electron donors and acceptors, and, in anticipation of challenges within the module electron transport, balance in the native quinone / quinol pool as well as NAD(P) / NAD(P)H pools, referred to as the redox balance. E.coli is quite the versatile model organism in its use of a broad scope of carbon sources and, depending on the source, might grow aerobically or anaerobically. Carbon flux thus strongly depends on growth conditions. On glucose and under aerobic conditions, glycolysis followed by runs of TCA will be the main pathway for generation of ATP. Anaerobic growth on glucose will lead to fermentation into, amongst others, acetate and ethanol.
Native electron transport chain(s) in E.coli
The electron transport chain in a range of organisms, including E. coli , comprises an enzymatic series of electron donors and acceptors in membrane-bound complexes situated in the mitochondrial inner membrane. Each electron donor passes electrons to an electronegative acceptor. This reduced acceptor donates the electrons to an following acceptor which is even more electronegative, a process that continues down the series until, in aerobic cultures, electrons are passed to oxygen, the most electronegative and terminal electron acceptor in the chain. The released energy from the transfer of electrons from donor to acceptor is used to generate a proton gradient across the mitochondrial membrane by the pumping of protons into the intermembrane space, which produces a thermodynamic state that has the potential to do work. This process is termed oxidative phosphorylation: ADP is phosphorylated to ATP using the energy of hydrogen oxidation in consecutive steps. A percentage of electrons do not complete the series and directly leak to oxygen. Bacteria can use a number of different electron donors, dehydrogenases, oxidases and reductases, and several different electron acceptors resulting in multiple electron transport chains operating simultaneously.
Competing sinks for electrons in E. coli where described as follows by Jensen  “The bacterium always needs the process of oxidation involving the respiratory chain and usage of a quinone-pool to stay alive. To make this work, we need to somehow create a division between the respiratory chain which provides the electrons and the respiratory chain that provides the cell with energy.“ How can or should we integrate or separate electron transfer- and respiratory pathways? First thoughts on this matter included the possible physical separation of the processes oxidative phosphorylation and extracellular electron transport, the saturation of the natural ‘stock’, or addition of an inhibitor of ATP-synthase. Are changes in relative flows able to be made, and if so, what is the impact on growth for one and EET on another level? Feasible option with which we continued was anaerobic growth in order to avoid oxygen pulling electrons through the native glycolysis followed by TCA and processed via oxidative phosphorylation.
Relevant basics of carbon metabolism
As mentioned in previous sections, native electron transport chains are present in Escherichia coli and form an interesting sidetrack regarding electron transfer. On a different level, implementing extracellular electron transport in model organism E. coli calls for a conditional electron acceptor electron transfer should be possible, however, the potential should be such in order for the compound to be able to ‘pull’ and to be reduced. Implementing the system in which growth as well as transfer is made possible is followed by creation of a system or device in which an electrode functions as acceptor and electrons are shuttled out of the cell in order for extracellular electron transfer to take place. Route of choice should, at that point, be the modified pathway of S. oneidensis implemented in E. coli . Control of operon expression regarding enzymes functional in carbon metabolism is exerted at the transcriptional level in response to the availability of (amongst others) the electron acceptors oxygen, fumarate, and nitrate. Oxygen is the preferred electron acceptor and represses the terminal reductases of anaerobic respiration. Energy conservation is maximal with oxygen and lower with, for example, fumarate. By this regulation pathways with high ATP or growth yields are favored. Oxygen, however is (too) strong an acceptor and, theoretically, will not make transport via routes that pose less promising redox potentials possible. Cultivation must thus be anaerobic. Thought experiments included fumarate as an electron acceptor, however, not only the potential is of relevance. Using fumarate, for example, will result in a change in pH. The formation of potentially toxic intermediates will have to be taken into account. Also, if there is a route for E.coli to reduce from a previous accceptor, it will do so and shift metabolism towards the original ‘waste’ product .
Several optional routes for generation of ATP and reducing factors have been considered. For example, glucose consumed anaerobically as a source of carbon would be able to generate ATP and necessary cofactors excluding the implemented electron transport system. Questions posed with respect to pyruvate utilization, generating ethanol, acetyl-CoA (incl. ATP) and fumarate, are, amongst others, whether there are transporters present in order to relief the cell from reaction products. What determines the conversion of pyruvate to acetyl-CoA when sinks are changed for EET purposes, there not seemingly being theoretical reasons for respiring it to carbon dioxide? In general, when looking into carbon source utilization, one would have to prevent alternative routes of generation of ATP. This includes prevention of substrate phosphorylation resulting in the formation of adenosine triphosphate (ATP) by the direct transfer of a phosphate group to adenosine diphosphate (ADP) from a reactive intermediate. A carbon source is needed in which substrate phosphorylation is not possible in order to prevent generation of ATP via this route. Glycerol, for example, is a potential source of carbon. However, there is a possibility of substrate phosphorylation from glycerol to pyruvate. Summarizing, in order to choose a feasible source of carbon, it is of importance to check possibilities regarding metabolism where substrate phosphorylation is not an option. Being quite the versatile organism, any fermentation pathway from which the organism is able to achieve a redox balance can replace the indigenous fermentation pathway(s) of E. coli . Jensen mentioned having specifically avoided carbon sources that provide types of growth other than anaerobic respiration (i.e. anaerobic fermentation), although she also points to the fact that "(..) the assumption that fermentation will siphon electrons away from the electron conduit has not been proven one could argue that a combination of fermentation and respiration could better support growth, and thus increase current out of the cell. Although I have done a series of experiments to determine what carbon source works best in our engineered E. coli strains, experiments using a mixture of these carbon sources with a fermentable sugar, such as glucose, may provide valuable insight into what impact fermentation has on anode reduction." .
Utilization of lactate as sole carbon source
E. coli, a facultative anaerobe, carries out mixed-acid fermentation of glucose in which the principal products are formate, acetate, D-lactate, succinate, and ethanol. During anaerobic growth, D-lactate is produced in order to recycle NADH produced by glycolysis. Cofactor NAD+ is generated, lactate secreted, the net supply per glucose being 2 ATP. This points to a first major issue in growth on lactate [5, 6]. Enzymes involved in the conversion of lactate to pyruvate and vice versa are LldD, specific for D-lactate, soluble LdhA, an NAD-linked fermentative enzyme and Dld, a membrane-associated respiratory enzyme. Formation of D(-)lactate from pyruvate catalyzed by D-LDH is most likely unidirectional [5, 6], which could mean pyruvate as a product in use of lactate could be (re)metabolized to lactate. Use of L-lactate and stimulation of E.coli to generate pyruvate in an enzymatic reaction using L-lactate specific L-LDH could pose an interesting solution to this potential problem. As Jensen reports, to date, increasing the number of conduits by transcriptionally upregulating mtrCAB has never increased iron oxide or electrode reduction, and it has not yet been determined why. As a possible explanation Jensen proposes that lactate oxidation by lactate dehydrogenase is rate limiting, inherently limiting the number of electrons delivered to the conduit.
In conversion of lactate to pyruvate, generation of ATP will be confined to a minimum and will thus affect growth. Aerobic growth on glucose followed by anaerobic generation of electron transport on lactate seems, summarizing, most feasible in development of a system in which extracellular electron transport is functional. Due to exposure to oxygen, enzymes functional in the tricarboxylic acid cycle (TCA cycle) will be expressed.
Redoxpotentials of elements central in electron transfer
Proteins of which the extracellular electron transport chain consists can be classified, in part, as cytochromes. Cytochromes are proteins containing one or several groups of heme, a porphyrrin structure able to bind iron. The basics for electron transfer are in fact formed by the redoxpotentials of the relevant compounds, the consecutive intracellular shuttles shifting electrons from the original electron donor. the carbon source. eventually towards a terminal electron acceptor, being the counterelectrode. Troubling are the redoxpotentials of several consecutive cytochromes, starting with CymA and / or native NapC. If NADH is not reduced, it will build up in the cell, as proposed in the section Modeling: Flux Balance Analysis of the EET Module and will form a bottleneck in generation of EET.
CymA and NapC carry several heme groups, of which the spin states of bound iron determine the redoxpotentials, that can vary considerably and will thus determine to what extent reduction (i.e. transport) takes place. It must also be mentioned that the transfer of electrons in vivo is, to a certain extent, determined by the surroundings of the protein considered as well as the states of, amongst others, redox poules and might thus be quite different than potentials determined in vitro .
Challenges and follow-up in generation of extracellular electron transport
Major concept of interest is to what extent generation of ATP for growth might take place, however, it must be considered if that is the actual objective – an in-field plug-and-play biosensor used in, for example, the laboratory, might be considered a one-use-only device in that case, it is of less importance to have proliferating cells. It might also be of interest to consider the possibility that once the EET has been saturated, the culture can be shifted towards aerobe glucose metabolism in order to get rid of the overflow of NADH. As the setup for measurements of current has been successfully implemented by this team, experiments might be executed in order to determine carbon sources maximizing EET. Also, in order to get a grip on use of D- and / or L-lactate, media could be measured after growth for the ratio of these isomers. FBA is per definition steady state closed experimental system no growth or flux. How can or should we integrate or separate electron transfer- and respiratory pathways? Could we separate these processes physically, or saturate the natural ‘stock’, or grow anaerobically?
Determination of bottlenecks must be continued via carefully designed experiments. Are there, for instance, alternatives to NapC and / or can we increase NapC transcription? What is the impact of use of the native versus an engineered cytochrome C maturation system? Is there an influence of heme availability on the system? Is it possible and useful to increase the amount of type-II secretion systems present? Can we optimize transport and conductivity by including electron shuttles or mediators, ie. riboflavins? (possible) native transmembrane electron transport E.coli.
An interesting final thought centers on redox potentials. As mentioned in the section ‘Redoxpotentials of elements central in electron transfer’, these potentials, resulting from differing spin states of iron, might pose a problem in transfer of electrons. Moving to a whole different section of Chemical Biology, redox potentials might in fact be adjusted. Reactivity of the iron center in heme depends on the coordination of iron by its ligands. Ligand chemistry, changing in the first coordination sphere, could decrease the overall potential of (for instance) CymA. At this point in time, this type of chemistry is not relevant for iGEM 2014. In a near future, however, it may very well be.
Final Electron Acceptor
That substance which receives the terminal waste product of cellular respiration.
The most common of final electron acceptors is molecular oxygen, O2, which combines with the spent electrons of cellular respiration, along with protons, to generate what is known as metabolic water. We breathe in to supply the oxygen necessary to keep cellular respiration going. Without oxygen the electrons in the electron transport chain have nowhere to go, so it backs up the system, shutting down the "aerobic" means of generating ATP.
There in addition exists a whole diversity of electron acceptors that are used in what is known as anaerobic respiration. Anaerobic respiration is used by numerous bacterial types, particularly prokaryotes that can be referred to as chemolithotrophs or "rock eaters". These non-oxygen final electron acceptors generally require higher energy electrons than oxygen, resulting in less energy extraction (proton pumping) than can be achieved by anaerobic electron transport chains.
Discussion and Future Directions
Instead of a hierarchal regulation of the respiration enzymes by a large number of transcription factors as is seen in E. coli and other bacteria (Table 1 Unden and Bongaerts, 1997 Arai, 2011 Hartig and Jahn, 2012), Campylobacterota possess a less sophisticated regulation of their branched electron transport chains. Adaptation to a host in a number of Campylobacterota has led to the loss many genes including metabolic regulators genes as the host provides a steady predictable supply of energy substrates (Moran, 2002 Koskiniemi et al., 2012). Instead of using multiple global and local regulatory proteins, the branched electron transport chain in Campylobacterota is subjected to more global cues from the environment. This is in line with the complexity of respiratory routes that correlates with the lifestyle of the Campylobacterota (Figure 1, Table 1). The respiratory behavior of these organisms can thus make us understand more about a bacterium lifestyle and potentially why, how, and when a bacterium becomes pathogenic.
There is little conservation between the regulatory proteins of the Campylobacterota, which resembles the high mutagenic and evolutionary rate of this phylum (Porcelli et al., 2013). Nevertheless, two main strategies of electron transport chain regulation can be distinguished: first, the repression of reductases that are lower on the redox hierarchical ladder and second, the substrate dependent induction of a specific reductase. Campylobacterota seem to rely mostly on the ladder mechanism. In the absence of the preferred electron acceptor, all other reductases seem to be expressed, irrespective of the presence of their cognate substrates. As a result, co-respiration of several chemical energy sources is likely to be a common event in Campylobacterota (Lorenzen et al., 1993 Weingarten et al., 2008 Dahle et al., 2013 Goris et al., 2015) a phenomenon also observed in some other bacteria (Fuchs et al., 2007 Chen and Strous, 2013), indicating that this represents an evolutionary beneficial method to efficiently adapt to environmental electron acceptors. There are observations of species prioritizing a fast growth rate over a higher growth yield (Lele and Watve, 2014). This behavior indicates that respiratory substrates are probably only temporarily available, and fast growth burst are the best strategy to gain an advantage over competing microorganisms (Foster et al., 2017). Another remarkable similarity is the conservation of formate and hydrogen respiratory enzymes in Campylobacterota. These donors, with low redox potentials are also implicated as essential for survival under certain (anoxic) conditions.
It is clear from this report that more data are needed, especially from the free-living Campylobacterota, to get a deeper insight how these bacteria regulate their electron transport chains. Several fundamental questions are still unanswered, such as what are the exact signals and mechanism that these bacteria use to adapt to the environment. There is a clear link between chemotaxis and respiration, since many chemoattractants are metabolic substrates and bacteria often cumulate inside optimal respiratory zones, but how they are mechanistically and molecularly linked is not known. Transcriptomics, proteomics, and metabolomics data obtained by growing these bacteria and appropriate derived mutants in the presence of different electron acceptors/donors are needed to further develop our understanding of the mechanisms used to regulate the electron transport chain in Campylobacterota. However regulation of the electron transport chain by more global cues from the environment and co-respiration are mechanism that play an important role in Campylobacterota and distinguish them from other bacteria.
Which is the final electron acceptor in the electron transport chain of cellular respiration? NADH FADH2 oxygen carbon dioxide
The answer is Oxygen .. so the name "oxidative phosphorelaton".
This potential is then used to drive ATP synthase and produce ATP from ADP and a phosphate group. Biology textbooks often state that 38 ATP molecules can be made per oxidised glucose molecule during cellular respiration (2 from glycolysis, 2 from the Krebs cycle, and about 34 from the electron transport system).
The correct answer would be:
Neither of these response options accurately features the anaerobic electron transport chain.
The anaerobic respiration system vibrated by an electron transport chain is a mechanism that anaerobic bacteria have to maintain their respiration.
This mechanism does not require oxygen in the atmosphere, that is why it is said to be an anaerobic mechanism.
Bacteria do not all need oxygen in the environment to live, some need that oxygen is not exactly present (strict anaerobes) or that it is at low partial pressures (facultative anaerobes).
This mechanism is very characteristic in its location since it is located in the inner membrane of the mitochondria, that is why it will decide to indicate that option as the correct one.
QUESTION 5 Which of the following is a product of the general photosynthesis equation? water oxygen carbon dioxide none of the above
During the process of photosynthesis, carbon dioxide and water are transformed into glucose and oxygen.
This reaction occurs when sunlight energy transforms six carbon dioxide molecules and twelve water molecules into one glucose molecule, six oxygen gas molecules and six water molecules.
Photosynthesis products are substances formed from the result of a chemical reaction, where reagents are broken down and rearranged.
Carbon dioxide and water are the reactants in photosynthesis and glucose, oxygen and water are the products.
Hai there :3 I'm planning to study chemical engineering.
Question related to Biochemistry (Photosynthesis & Cellular Respiration)
1. Chemiosmosis. In the process of chemiosmosis, specific enzymes (such as ATP synthase) create ATP. Hydrogen ions go from a higher proton concentration to a lower one, which is why it's called chemio"osmosis"
2. Electron Transport Chain (ETC). The name says it all. Simply explained, electrons are transported and transferred in the mitochondrial membrane.
3. Oxygen. O2, the diatomic molecule, is essential in respiration. In the final stage of respiration, at the near end of the electron transport chain, oxygen accepts protons to become water. Cells use O2 during oxidative phosphorylation.
4. NADPH. I remember learning what this acronym means by heart. Nicotinamide Adenine Dinucleotide Phosphate Hydrogen. NADPH is essential in photosynthesis as a typical coenzyme in the reduction of chemical reactions.
C. Coupled reactions establish an electrochemical gradient across a membrane.
During cellular respiration in mitochondria, and during photosynthesis in chloroplasts, the electron transport chain requires a proton gradient to pump protons across the membrane by active transport. Protons flow back across the membrane by facilitated diffusion through ATP synthase, which utilizes them to phosphorylate ADP to ATP.This process of ATP synthesis by harnessing the elctrochemicaal gradient geenrated by the diffusion of protons across the biological membrane through ATP synthase is called chemiosmosis.
4 When ADP adds another phosphate, it becomes . ATP AMP ABP none of the above
ADP = adenosine diphosphate
ATP = adenosine triphosphate
so when another phosphate is added to ADP, it becomes ATP.
In fact, during the actual conversion, the product is ATP and AMP. ATP has one more phosphate, AMP has one less.
1) The electrons that travel down the electron transport chain come from the NADH and FADH2 molecules produced in the three previous stages of cellular respiration : glycolysis, pyruvate oxidation, and the citric acid cycle.
2) At the end of the electron transport chain is the Oxygen that will accept electrons and picks up protons to form water.
If the oxygen molecule is not there the electron transport chain will stop running, and ATP will no longer be produced.