Three major metabolisms:Chemoorganotrophic metabolism: Organisms are using organic carbons as a source of carbon and energy. In this strategy, organic compounds are oxidized and electrons are passed down an electron transport chain to create a proton motive force. The electron acceptors for this are oxygen, other compounds like elemental sulfur, nitrate, and organic electron acceptors like fumarate. Organisms use this as a source of electrons and as a source of biosynthesis
Suraj
Note
Chemolithotrophic metabolism: They use inorganic compounds as the electron donors. They use similar ETC and they generate a PMF that they use to make ATP. The electrons go through the same type of compounds that we see for the chemoorganotrophs. They have to have a different way of getting their carbon for biosynthesis and they do that by autotrophy so they fix CO2 and they biosythesize. So the inorganic compounds in addition to providing electrons for ETC, also have to provide electrons for CO2 reduction. So they have 2 jobs. In the organic case, they can just take the reduced carbon directly into biosynthesis. For the lithotrophs, they have to take the electrons and use it to reduce CO2 to the level of cellular carbon. How many electrons do you need to reduce CO2 to cellular carbon? --> 2. How much oxygen? --> 1. How many electrons does it take to go from CO2 to CH2O? --> Four electrons!
Suraj
Note
Phototrophic metabolism: You have light energy that is used to power the ETC to generate PMF to make ATP. You have 2 types of phototrophy: Photoheterotrophs: Carbon is coming from an organic comopound and you have photoautotrophs, carbon is coming from CO2 reduction.
3
Feel challenged? Convert equations to sentences. Review freshman chemistry. On the quiz, you will have to use these equations, but you will not have to remember them.
Suraj
Note
Chemiosmotic hypothesis is represented by this: Delta p which is the PMF or the electrochemical potential, is the difference of protons across the membrane. It is made of two components: Delta psi and the minus Z delta pH. Z is the constant. Delta pH is the change in the proton concentration across the membrane. In the chemisosmotic hypothesis, this relates directly to the change in free energy. How do we use this? Cells tend to maintain the internal pH which stays more constant than the environment does. The cells also try to keep delta p the same. It's usualy about -100 to about -200 mV. If you are keeping the internal pH the same, and you are changing the external pH, how do you compensate to keep the delta p about the same? --> You do it by changing the delta psi. You find that organisms can have very different ion gradients depending upon the growth circumstances but also depending upon the type of organism that they are. If you are an acidophile, you have a low pH outside the cell. the inside of the cell is about 6 which is close to neutral so your pH gradient is about 4-6. the delta p is going to be about the same as a neutrophilic organism, so whats going to happen to the delta psi? --> The cells are manipulating the ion component to keep the internal pH constant which is required to keep the proteins in the right form and also to keep the delta p constant, because this is what is actually being selected on
Suraj
Note
A proton is a monovalent cation. What's another ion that is abundant on earth? --> Sodium Do you think that organisms could also use sodium and would it function in generating a PMF? There are many organisms that do use sodium and they don't have a proton gradient. They have a sodium gradient. The PMF doesn't require a proton gradient. It could be any ion. Other ions could include chloride and potassium
Suraj
Note
The smaller the change in midpoint potential, the smaller the change in free energy Example: Delta E = midpoint potential of the acceptor minus the midpoint potential of the donor and that's +.353V. So you take the delta E and substitute it into the very bottom equation and you end up with a change in free energy of -68kJ/mol Why are most oxidation reduction reactions that we see in a cell involve 2 electron transfers? So why is succinate to fumarate oxidation or the reduction of fumaratte to form succinate a 2 electron reduction? --> Because most of these reactions involve forming/losing a covalent bond and there are 2 electrons in covalent bonds in carbon compounds. It's not true for all of the oxidation reduction reactions in biology. For example, iron just involves 1 electron in covalent bonds
4
How large must the DE be to make ATP?
[no calculators, please]
Suraj
Note
Delta G = -nFDelta E 50 = 200 Delta E So delta E = 250 mV The midpoint potential: Think of it as a chemical reaction related to the equilibrium constant of a chemical reaction. It's another way of writing the equilibrium constant for a chemical reaction. It's very useful becuase its a nice shorthand when you are measuring certian kinds of measurements in electrochemistry.
5
Figure 10.10
Mitochondria and Paracoccus
electron transport
-200 mV +35 mV
+50 mV
+385 mV
Eo’ of carriers (mV): NADH, -320; FADH, -220; succinate, +31; Q, +100; cyt c +230; H2O, +820
Suraj
Note
intermembrane should be the periplasmic space in Paracoccus The mitochondria probably evolved from a bacterium that was close to Paracoccus and that's why they are similar and behave identically and function the same way. The mitochondrial ETC is just a bacterial ETC put into eurkaryotes. Because this is so important, you SHOULD JUST MEMORIZE THIS AND BE ABLE TO DRAW IT. REPRODUCE THIS IN DETAIL
Suraj
Note
Also, you should be able to know the TCA cycle and know the structures and compounds because it's beautiful!So basically, the electron transport chain generates a PMF across the membrane and it has 3 places where protons are generated. NADH dehydrogenease oxidizes NADH and passes the electrons to a flavin mononucleotide to an iron sulfur cluster and eventually to quinones. Complex II, sometimes called succinate dehydrogenate, oxidizes succinate and reduces flavin and passes the electrons onto a iron sulfur cluster inside it. Flavin can accept 2 electrons and can pass electrons one at a time. The electrons then go to the reduced quinones. The reduced quinones then get oxidized by cytochrome b,c complex and this is Complex 3 and it is also the second coupling site for ETC. This is the second set of proton pumps. From here, this reduces cytochrome C and this is a protein that is mobile and it is on the outside of the cell and it can move to cytochrome oxidase which is coupling site 3 and complex 4. It is an oxidase so it reduces molecular oxygen to form water and it also translocates protons across the membrane. Complex just reduces the quinones. The oxidation of quinones by cytochrom b, c, complex
Suraj
Note
You take NADH and oxidize it and the electrons are transferred and you end up reducing oxygen to water and you generate a PMF and the ATP synthase uses that to make ATP. Complex 1 is also called NADH dehydrogenase because it oxidizes NADH. The midpoint potential of the complex is about -200 mV. NADH donates 2 electrons to flavin and then the flavin donates 1 electron twice to the iron sulfur cluster and they are donated to quinones. NADH has a midpoint potential of -320 mV. We could oxidize NADH and reduce succinate to fumarate and we would still make ATP.
Suraj
Note
Succinate can be oxidized and the electrons go to flavin (FAD) and then it goes to the differenet iron sulfur clusters 1 electron at a time and then it goes to cytochromes which are also 1 electron carriers. From there, a pool of quinones are reduced and now you have QH2 dissovled in the membrane. These reactions are occruing at the lipid bilayer. Quinone is oxidized by the cytochrome b, c complex. The general complex has a midpoint potential of about +35 mV. Could you generate ATP from the oxidation of succinate to the reduciton of this complex? --> Not really. the change in midpoint potential is too small. The boudn cytochrome C transfers the electrons to this free cytochrome C and it moves back and forth between the b,c coplex and the cytochrome oxidase in complex 4. Now going from succinate to the cytochrome oxiase in complex 4, you have a midpoint potential of about 300 mV, you could generate a PMF here and thats what occurs at hte cytochrome b,c complex. These complexes all function in the oxidation of quinones and generation of PMF across the membrane. In the mitochondria, the last step (cytochrome C) is reduced and oxidized by cytochrome oxidase and this is cytochrome a3
Suraj
Note
In the quinones, the midpoint potential is also affected by the environment it is in. The midpoint potential of the pure compound does not necessarily predict accurately what is in a cell. Thus, these are just approximations. Also, changes in free energy dont' predict rates. All reactions are reversible. Changes in free energy predict end points so they predict concentrations of equilibrium. The last reaction takes cytochrome C at +230 and passes electrons to oxygen to make water at about +800 so this change is much greater than 250 mV and this translocates protons as well and you can make ATP.
6
Figure 10.13-Q cycle
Find the typo!
The only mechanism of H+ pumping that is really understood
Suraj
Note
Only mechanism of proton pumping that is actually understood. There is a typographical error on this figure. Here we are starting with QH2, which is the reduced form of quinone. It is going to donate electrons to the cytochrom b, c complex. one electron goes to reduce the iron sulfur cluster and the other electron goes to reduce cytochrome b. it forms the fully oxidized quinone Q and it also drops 2 protons on the outside of the membrane. They are not hydrogen atoms which would be a proton plus an electron. One electron goes to the iron sulfur cluster and then goes to cytochrome C. And this is going to be transferred to the oxidase. So we have removed one electron from the system and pumped 2 protons. The electron that goes to the cytochrome B comes around and reduceds a fully oxidized quinone to form a semiquinone. At the end of this reaction, you have oxidized one quinone but you have reduced one quinone half way and you have pumped 2 protons and moved one electron to cytochrome C. In the second half of the reaction, you take a second fully reduced quinone and you oxidize it and does the same thing except that you are adding 2H to the semi quinone and you get QH2. So that's the typo. So, you take two protons out and you take protons in as well. You are still taking electron. So, you end up translocating 4 protons for 2 electrons that pass through the system.
7
Figure 10.15 –What types of energy
conversions are catalyzed by the ATP synthase?
Suraj
Note
The ETC generates the PMF, but it's the ATP synthase that utilizes it. this is a cartoon version. there are 2 components. there's the F0 component, which is in the cytoplasmic membrane of bacteria, and there is the F1 component, which is hydrophilic and sits on top of F0. In response to the PMF, this F1 component actually spins around and PMF will cause the F1 to spin around. In the cell, it might be the F0 spinning around, but it doesn't matter. This is a diagram of what seems to be happening during the F1 relative to F0. This is the gamma subunit and it is a trimer and there are 3 binding sites for ATP, BE is empty. TP has ATP bound and one is DP, which is ADP bound. When it turns and moves around, the conformation of the BE to BHC and now it can bind ADP and inorganic phosphate. This is a low energy binding. As the subunit moves around, the ATP is ejected and the ADP here gets converted to ATP. The ADP on the right in the 3rd figure is in an high energy state and there is very little energy needed to convert that state to ATP. When that happens, the ATP comes off and you have the same 3 subunits that you started with, but now they are in the different ones.
8
What types of energy conversions are
catalyzed by the ATP synthase?
Conversion of ‘chemiosmotic energy’, ie. the
energy stored in a gradient across a
membrane, into ‘conformational energy’ , ie.
the energy stored in the structure of a
macromolecule, as well as the conversion of
conformational energy into ‘covalent bond
energy’, ie. in a small molecule such as ATP
Suraj
Note
Energy conversions: PMF is converted to conformational changes and it's also been converted to kinetic energy (rotation). The chemiosmotic energy (energy in the gradient) is converted to conformational energy and then it converts that to covalent bond energy. Another example of converting chemiosmotic energy to kinetic energy is the rotation of the flagella. An example of converting conformational energy into covalent bond energy is muscle cells (myosin). You can take ATP (chemical) and convert into mechanical (kinetic) energy.
9
Variations in respiration typical among
prokaryotes
1. Aerobic respiration- modifications of the
electron transport chain are typical; especially
common are a wide variety of terminal
oxidases
2. Anaerobic respiration- use of an electron
acceptor other than O2.
3. Different entry points into electron transport
chain depends upon Eo’ of electron donor-
lithotrophy
Suraj
Note
3. Different electron donors than NADH or use donors with higher midpoint potential and that may cause variations in respiration
10
Figure 10.12 Electron Transport Chain of E. coli Branched pathway Different cytochromes used than in the mitochondria Notice different numbers of protons are pumped
Suraj
Note
2 oxidases and they are expressed under different growth conditions. Under high aeration and in log phase growth, it makes this oxidase (copper containing oxidase) and it's a proton pump; when electrons come from quinone, it pumps protons and reduces oxygen to water. Cytochromes are named by the absorbance spectrum and 542 is the absorption maxima for this cytochorme and it is a conventient way of telling cytochromes apart. Under low oxidation in the stationary phase, you oxidize the quinones reduce oxygen. It doesn't pump protons and doesn't do anything for the cell. Under low oxygen, we have very low amounts of the substrate and the reaction is not that favorable and it is very hard to couple it to proton movement. You can still pump protons in the ETC earlier at the NADH dehydrognease.
11
Figure 10.9- Tower of Power In anaerobic respiration, different components of the electron transport chain are used depending upon the Eo’ of the acceptor
Suraj
Note
At the top of are the better electron donors. So glucose is a good eletron donor. Oxidized species is first and reduced species is last. These are all reversible reactions and we have the midpoint potential of them. At the bottom, we have oxygen and water. This reaction has a very positive midpoint potential of 815 mV. Bacteria if they are not using oxygen as their electron acceptor, they might be using many of these things as their electron acceptors. That's why midpoint potential of electron acceptor is very important in ETC
12
Denitrification- nitrate is the electron acceptor. Which components of the electron transport chain are used?
-200 mV +35 mV
+50 mV
+385 mV
Suraj
Note
Forming NADH and you want to oxidize NADH. But instead of using oxygen as the electron acceptor, you are using nitrogen. What component of ETC would you use? Complex 1,2,3, or 4? --> You are taking electrons from NADH and component 4 works at the same midpoint potential as nitrate. If you pass electrons from component 4, you are not going to be able to pump protons because the change in midpoint potential is close to 0 So no good if you go through 4. but if you go through 1, you can pump protons, reduce quinones, and you go through component 3 and go through Q cycle and reduce cytochrome C. instead of going to component 4 where you can't pump protons anyway, you go through other proteins that will reduce proteins. the point is that using these other electron acceptors that are more electronegative than oxygen, you end up dumping/cutting off the ETC, you use components that are more electronegative and don't use component 4
13
Electron transport chain in Paracoccus during denitrification. Denitrification occurs in a sequential series of reactions Reactions Eo’ (Mv) NO3
-/NO2- +430
NO2- /NO +360
NO/N2O +1180 N2O/N2 +1360 NO3
- is nitrate NO2
- is nitrite NO is nitric oxide N2O is nitrous oxide
Suraj
Note
this is the ETC for Paracoccus on bottom and top is ETC for Paracoccus during denitrification. It takes cytochrome C and instead of going to cytochrome C oxidase, it goes to a protein that can reduce the different nitrogen species that are formed during denitrification.
Suraj
Note
In this case, the NADH dehydrogenase reduces the quinones and goes to the nitrate reductase which reduces nitrate to nitrite and the nitrite is reduced by cytochrome C to form nitric oxide. Or the cytochrome C can go to another protein which reduces the nitrous oxide to N2 or it can reduce NO to nitric oxide. The reduction of nitric oxide and nitrous oxide are not coupled to proton pumping even though the midpoint potentials are very favorable. we just don't know why!
14
Sulfur reduction- sulfur is the electron acceptor. Which components of the electron transport chain are used?
-200 mV +35 mV
+50 mV
+385 mV
Suraj
Note
sulfur is an electron acceptor and is reduced to hydrogen sulfide. Midpoint potential for this reaction is -270 mV. So, if you are starting with NADH, its about -320 mV, so what do you think we use? complex 1,2,3, or 4? We don't know. Probably NADH dehydrogenase
15
Table 10.1- Eo’ of acceptors +820 mV
+360 mV
+740 mV (N2)
-220 mV
-240 mV -280 mV -270 mV
+770 mV but ~+200 mV at pH 7
+140 mV
+475 mV
+ 33 mV
Suraj
Note
HAsO4 (2minus): Oxidation state is +5 and oxidation state of HAsO2 is +3 so you add 2 electrons.
16
Why are prokaryotic electron transport chains so diverse?
Suraj
Note
For aerobic respiration, you can have branch ETC. For anearobic, same thing, and it can be made in many different components and why is that true for prokaryotes and not eukaryote? In anaerobic respiration, you can oxidize NADH and where you take it out corresponds to which complex you use. In denitrification, organisms don't use Complex 4 because midpoint potential is too close to the electron acceptor and they can't make ATP.
17
Figure 10.9- Tower of Power For lithotrophs, different entry points into electron transport chain depend upon Eo’ of electron donor
Suraj
Note
Nitrate and Nitrite can also be electron donors along with being electron acceptors.
18
Nitrification- ammonia and nitrite are the electron donors. Which components of the electron transport chain are used?
-200 mV +35 mV
+50 mV
+385 mV
Suraj
Note
Here again is the ETC in Paracocccus. The midpoint potential of complex 1 is about -200 mV and the midpoint potential of complex 3 is +35 mV and the midpoint potential of complex 4 is about +400 mV. Which one of them can be used ass an electron acceptor for a favorable reaction rather than oxygen? Which would you use nitrite as the electron donor and oxygen as the electron acceptor and the reaction is favorable and you could generate a PMF? --> Complex 4. You would think that you would oxidize nitrate or oxidize ammonia and you pass the electrons through complex 4, you can reduce oxygen to water. If you are reducing oxygen to water, how many electrons would you need? This is the 2 electron reduction when you go from oxygen to water. If you have oxygen going to water and you are using nitrite as the electron donor, how many nitrites to half oxygens? This is a two lectron reduction from Nitrite to nitrate and 2 electron reduction to go from oxygen to water
19
Figure 10.26- Nitrobacter electron transport chain. Reverse electron transport is used to reduce NAD for biosynthesis.
Suraj
Note
The enzyme that oxidizes nitrite to form nitrate generates electrons tocytochrome C in Nitrobacter and these are oxidized by the cytochrom oxidase and thats how this organism generates PMF and that's how it makes ATP. The organism also has an additional problem. In addition to making ATP, you need NADH to grown and reduce CO2. How does it do that? in this case, it takes an electron and reduces another cytochrom C and this goes into the cytochrome b,c complex and this reaction is very unfavorable. How do the cell use it? They use the PMF to drive this in reverse. Some of it is used to make ATP but some is also used to drive the reduction of ubiquinone (coenzme Q). Likewise, coenzyme Q is used to reduce NAD to NADH and this is also an unfavorable reaction. How do cells get it to work? They use the PMF to drive the reaction in reverse. In lithotrophs, you are adding electrons in the middle and running to end to reduce oxygen and you are also adding electrons in reverse to generate reduced-pyridine nucleotides
20
Tables 10.3/10.4
Homework: Calculate the changes in midpoint potential (Eo’) from the changes in free energy. Do your values make sense?
Suraj
Note
These are representative chemolithotrophs, organisms that use electron donors other than glucose or carbon. They can use nitrate as the electron acceptor and you can reduce the nitrate to N2 and you oxidize the sulfur compounds to sulfate. What happens if all you have are oxidation reduction reactions? See next slide
21
Photosynthesis Chlorophyll-based photosynthesis uses light energy to make good electron donors from poor electron donors. It then uses the electron transport chain to generate a PMF or reduce NAD(P).
Bacteriorhodopsin-based photosynthesis uses light energy to generate a PMF directly. The major pigment is retinal instead of chlorophyll. Electron transport is not involved.
Suraj
Note
Without photosynthesis, what happens? what's the ultimate source of energy? --> You can oxidize organic carbon to CO2 and we can go back by doing photosynthesis. In photosynthesis, we can take a crummy electron donor (water) into a good electron donor. If you do't have photosynthesiss going on, everything gets oxidized! and thats not good. How do cells grow? --> They grow by coupling growth to chemical reactions so if there is no chemical reactions going on, there is no growth. There is one other energy source on Earth other than the sun. If you go to the deep sea, you have these hypothermal vents and you have these reduced compounds coming out and these are the only sources of electron donors in the absence of photosynthesis. Pigment retinal absorbs light. it cause s a conformational chagne in the protein and the protons are pumpted across the membrane and it is a light driven pump. in chlorophyll based photosyntheses,, light energy is used to make a curmmy electeron donor into a good donor. Know Table 10.6 (basics of it)
22
Chlorophyll based photosynthesis: Light is captured by the light-harvesting complex, transferred to the reaction center, which is the site of the photochemical event. Extensive diversity of the LHC in prokaryotes allows for capturing light of a variety of wavelengths and conditions. In contrast, the RCs are more conserved- there being only two types, RCI and RCII. The LHC is the site of photon capture, ie. the conversion of light energy to the excited state of the pigment molecules. The RC is the site of the photochemical event, ie. energy conversion from the excited state of the pigment molecules to covalent bond energy in generation of a strong reductant and strong oxidant. A ‘charge separation’ Always associated with membranes.
Suraj
Note
The LHC is the part that deals with the outside and how the photosystesm interacts with the environment. imagine LHC captures a photon, absorbs light energy, and converts light energy to excited state. Charge separation is when you have a strong reductant and a strong oxidant
23
Organization of the phototrophic apparatus in different groups of phototrophic bacteria. OM = outer membrane, CW = cell wall, CM = cytoplasmic membrane, RC = reaction center, LHC = light-harvesting complex. Question marks indicate that the organization of the cell envelope and the organization of the photosynthetic apparatus in Heliothrix oregonensis is not exactly known. From: JÖÖRG OVERMANN and FERRAU GARCIA-PICHEL (2003) The Phototrophic Way of Life. M. Dworkin (ed.) The Prokaryotes, 3rd edition, available on-line.
24
Photosynthetic apparatus in the cyanobacteria. Cyt = cytochrome; P840 and P870 reaction center special pair = primary electron donor; B800, B850, B875 = bacteriochlorophyll molecules bound to light-harvesting complexes II and I; A 0 = primary electron acceptor in green sulfur bacteria = Chl a; A 1 = secondary electron acceptor in green sulfur bacteria = menaquinone; Q A, Q B = ubiquinone; FX, FA, FB = FeS-clusters bound to the reaction center; Fd = ferredoxin; FMO = Fenna-Matthews-Olson protein; FNR = ferredoxin NADP+ reductase; PQ = plastoquinone; PC = plastocyanin; PS = photosystem. From: OVERMANN and GARCIA PICHEL (2003) The Phototrophic Way of Life. M. Dworkin (ed.) The Prokaryotes, 3rd edition, available on-line.
Phycobilisome- primary LHC to both RCI and RCII
Suraj
Note
Generating a strong reductant which donates electrons and a strong oxidant which accepts electrons. Instead of donating electrons to cytochrome C, it donates to copper Top half is cytoplasm and bottom is periplasm When you reduce quinone here, it is a homolog of the cytochrome b,c complex called cytochrome b,f. And istead of donating electrons to cytochrome c, it donates it to a copper protein called plastocyanin and then donates to another reaction center 1.
25
Photosynthesis in the cyanobacteria is very similar to that in the chloroplast. Major differences are in the LHC. Otherwise, the both have two RCs and generate a PMF for ATP biosynthesis and NADPH for CO2 reduction.
Why are two RCs necessary to reduce NADP with H2O as the electron donor?
Suraj
Note
When P680 is excited by light, it reduces Pheo. if you have a chlorophyl without magnesium, you have pheophydin. it becomes an electron donor and donates an electron to QA. Purple bacteria absorb towards the red. There are 2 quinones in this molecule, Chemical reactions fairly slowly. Once the Qb is reduced, it leaves the complex and diffuses into the membrane. The plastiquinone (QB) is then oxidized by the BC complex. Iron sulfur crystals play a very important role in the electron transport chain
Suraj
Note
RCs = Reaction centers --> Most of the energy is lost and the cell doesn't capture all the energy --it only captures half of it. If you take two things and put them in series and you add an additional jolt of energy, you can reduce Pherodoxin.
26
Oxygenic photosynthesis appear to have evolved about 2.3 billion years ago, corresponding to the ‘Great Oxidation Event’ of the earth’s atmosphere. Microbial life is at least 3.8 billion years old. Why do you think it took so long for oxygenic photosynthesis to evolve?
Suraj
Note
Not a lot of oxygen in the atmosphere Presumably, one of the reaction center evolved first and then duplicated and then the second evolved. Then you have to have an enzyme that split water and that's why it took so long. It is a unique enzyme. Also, the organisms themselves have to be resistant to oxygen. Aerobic organisms had to learn how to respire first themselves in order to start doing oxygenic photosynthesis. It's really a matter of what happens to itself than what happens to the community. Another theory is that the earth was constantly bombarded by asteroids and meteors that created a smog in the atmosphere that prevented the passage of light and light is very important for photosynthesis.
27
Photosynthetic apparatus in the purple bacteria. CM = cytoplasmic membrane; Cyt = cytochrome; P840 and P870 reaction center special pair = primary electron donor; B800, B850, B875 = bacteriochlorophyll molecules bound to light-harvesting complexes II and I; QA, QB = ubiquinone; FX, FA, FB = FeS-clusters bound to the reaction center; Fd = ferredoxin; From: OVERMANN and GARCIA-PICHEL (2003) The Phototrophic Way of Life. M. Dworkin (ed.) The Prokaryotes, 3rd edition, available on-line.
Suraj
Note
Reaction Center was crystallized from a purple bacterium and the structure was worked on in great ail. Diamonds are chlorophyll whereas the symbols below are hemes that look like a parallelogram. If you take a bacterium and grow it anaerobically in the light, it will be pumping protons through the cytochrome b, c complex
Suraj
Note
How do cells make NADH and how do they reduce NAD? they can use some of the ubiquinone and because it is a lousy electron donor, so they use the reverse electron transport and they use the NADH dehydrogenase to reduce NADH. The three circles are flavin. So it would be FMN. The cube looking thing below are the iron sulfur clusters. So, the NADH dehydrogenase has a flavin and two iron sulfur clusters.
28
Anoxygenic phototrophs use two ways to make NAD(P)H. In the green bacteria, NAD is reduced by ferredoxin produced by the photosynthetic electron transport chain. Electrons are returned to the reaction center by electron donors such as sulfide or thiosulfate. Noncyclic photosynthesis. In the purple bacteria, photosynthesis generates an PMF. Reverse electron transport then reduces NAD with poor electron donors such as succinate (but also sulfide and thiosulfate). Cyclic photosynthesis.
Suraj
Note
Half of the energy is lost in this rapid reaction. Now you make quinone and goes through the ETC and re-reduces the reaction center. OR you can take the electrons out and use reverse-electron flow to reduce NAD. How do you re-reduce the ETC? In the cyclic case, what happens if you take the quinone out and you use it to make NADH? You can oxidize reduced sulfur compounds and rereduce the quinones that way. You still need some sort of electron donor to re-reduce the quinones to provide the electrons for the reaction center.
Suraj
Note
Sulfur bacteria do something different. When they are excited, they go through iron sulfer clusters and has the option to reduce ubiquinone and going to the cytochrome B, C complex. It also has the option to reduce ferredoxin and then reduce NAD. How do you re-reduce the ETC? It takes the elelctrons from hydrogen sulfide or theosulfate to re-reduce the ETC. This is acyclic photosynthesis anoxygenic phosphatate and its using external electron donors to re-reduce the ETC Therefore, if it goes below to the left, it's cyclic and if it goes to the right, then its acyclic.
29
Heterotrophy- the dark side of life
• many different energy sources are funneled into common degradative pathways
• many pathways generate glucose or intermediates of the pathways used in glucose metabolism
• Using common degradative pathways greatly increases metabolic efficiency for generalist organisms- what might specialists do?
Suraj
Note
For chemical reactions to occur, you reduce CO2 to make a compound and you have to re-oxidize it and you have to have a cycle and heterotrophy is the dark side of life and it is the side that breaks down the organic compounds. one strategy is that many different energy sources are funneled into a degradetive pathway. Imagine that each sugar can have its own pathway for degradation. Thus, you need 100s of pathways and it would be very inefficient. Therefore, bacteria have some common degenerative pathways and they funnel different substrates into that pathway that greatly increases metabolic efficiency for generalists organisms. Specialists can be like bacteria that break down methane gas. These guys don't use other substrates. They are highly specialized for that kind of metabolism. Organisms use many different way to make a living. Specialists will adapt to growing on one thing but that thing is always abundant and always available.
30
Figure 10.2-Heterotrophy- the dark side of life many different energy sources are funneled into common degradative pathways Using common degradative pathways greatly increases metabolic efficiency for generalist organisms- what might specialists do?
Suraj
Note
Where do these things come from? Proteins come from the living matter of cells and about 50% of roganic matter is protein. Polysaccharides only comprise 5-10% of organic matter. Most polysaccharides such as cellulose come from wood. Then you have phospholipids from membranes and it's also a fairly small component of the cell. These are all broken down into their monomers and they all feed into the glycolytic pathways and the TCA cycle. Using common pathways increases their metabolic efficiency for a generalist organism
31
Figure 10.3- Prokaryotic cells are usually not compartmentalized, so catabolism and anabolism occur simultaneously in the cytoplasm. Amphibolic pathway: many of the reactions function in both catabolism and anabolism. A few enzymes that catalyze irreversible reactions serve as the ‘gate keepers’. How do you prevent a ‘futile cycle’? Name some common amphibolic pathways:
Suraj
Note
All enzymatic reactions are reversible. the reaction of glucose-6-phosphate to fructose-6-phosphate is irreversible. They are used during gluconeogenesis and glycosis. Some steps are irreversible. Pyruvate kinase only functions in one direction. In principle, all enzymatic reactions are reversible because enzymes work by lowering activation energy. They make it irreversible by adjusting the coenventrations of substrates and products.
Suraj
Note
What's the problem in amphibolic pathways? If both of these enzymes are active at the same time, both forward and reverse reactions are operating at the same time and it is a futile cycle. you are basically burning ATP and cell works to prevent this by 1) controlling substrates and intermediates and 2) uses other effectors to control which of these enzymes are active. These enzymes may only be active in the presence of a compound that makes them active.
Suraj
Note
Go through the next few slides and identify more examples of amphibolic pathways yourself!
32
Figure 10.4- Embden-Meyerhof Pathway. Most common pathway for glucose degradation to pyruvate. Look for the addition of phosphates to activate the sugar, oxidation steps to generate NADH, biosynthesis of high-energy molecules for making ATP by substrate-level phosphorylation
Suraj
Note
Read over this! Be familiar with them enough so you can identify the intermediates. This is the most common pathways in glucose degradation.
33
Summary of Embden-Meyerhof Pathway.
glucose + 2ADP + 2Pi + 2NAD+
2 pyruvate + 2ATP + 2NADH + 2H+
Note that 2 ATPs are invested in the activation and 4 ATPs are produced, for a net of 2 ATPs.
Suraj
Note
2 ATPs are invested to activate the glucose to make fructose-6-phosphate and you get 4 ATPs produced in the end along with 2 NADH. Anytime you oxidize glucose to pyruvate, you are going to generate 2 NADHs. Whats really different in the different pathways is the amount of ATPs.
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Figure 10.5- Entner-Duodoroff Pathway. Less common that EM pathway but still widely distributed in bacteria and archaea. Net yield per glucose molecule:
1 ATP 1 NADPH 1 NADH
Why use this pathway if the net yield of ATP is less?
Suraj
Note
Alternate pathway of sugar degradation. Only found in prokaryotes. Glucose-6-phosphate is oxidized to form 6-phosphogluconate. Then you remove water. The pathway going from Glyceraldehyde-3-phopshate to Pyruvate is the same as Ender-Myerhoff pathway.
Suraj
Note
Not sure. Fermentative organisms use Embder Meyerhoff pathway and aerobic organisms use Entner-duodoroff pathway. The reason being that aerobic organisms are not limited by ATP very often and they are really limited by nitrogen phosphate or the availabiality of sugars. Entner-Duodoroff selects for organisms that can utilize ATP. For an aerobe, it is just a measure of utilizing different types of sugars. Advantage of Entner-Duoderoff is their versatility to take in a wide variety of sugars
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Figure 10.6- Pentose phosphate Pathway. Can operate at same time as Embden-Meyerhof or Entner-Duodoroff pathways; an amphibolic pathway, esp. for ribose biosynthesis; Over all yield:
glucose-6-P + 12NADP+ + 7H2O
6CO2 + 12NADPH + 12H+ Pi
Note that EMP reactions generate 2 ATP + NADH per pyruvate made.
Suraj
Note
Can operate at the same time as Emden Myeoff and Entner Duoderoff pathways. It's another amphibolic pathways especially for ribose biosynthesis. Pentose phosphate pathway is invovled in regenerating the C6 phosphate from C5 phosphate. You are generating 2 C6 and 1 C3. This pathway is one common way for cells to make pentose. It's also a way for cells to metabolize pentose for polymers and you can make hexoses for biosynthesis. Why use it? --> The overall yield from glucose 6 phosphate is NADH and CO2 and it does not make ATP at all. The production of NADH for biosynthesis and sometimes the cell just doesn't need ATP and just needs NADPH. This pathway does some things differently than the Embden-Meyerof pathway.
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Why use the Pentose phosphate Pathway? 1. Production of NADPH for biosynthesis. 2. Production of ribose-5-phosphate for nucleic acid
biosynthesis and erythrose-4-phosphate for aromatic amino acid biosynthesis.
3. Yields some ATP. 4. Allows for catabolism of pentoses.
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Use your general knowledge to estimate (or guestimate): How many genes in a prokaryotic cell? How many proteins are present at any one time? How many enzymes are present at any one time? How many enzymes are active at any one time? How many of these enzymes catalyze reversible reactions? So what does it all mean?
Suraj
Note
1.) 20,000-30,000 genes in eukaryotes. So, in prokaryotes there are about 1,000-10,000 genes 2.) Half of proteins are probably made at one time. (~1,000 proteins) 3.) In bacteria, most proteins are enzymes. So, about 950 enzymes 4.) Most are active at one time. So about 950 active 5.) Most of them. 90% of them. What does it all mean? --> They are just gross simplifications! Think of it as a mess
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Figure 10.7- Tricarboxylic acid cycle. The best pathway on earth. Probably the most widely distributed pathway in prokaryotes. yield per acetyl-CoA molecule:
1 GTP 3 NADH 1 FADH2
2 CO2
Also a major source of intermediates for amino acid biosynthesis.
Suraj
Note
Generally, the Krebs cycle is a way of degrading acetyl-CoA. For organisms that aren't growing on sugar, they are also producing intermediates for gluconeognesis. Know all this pathway including structures and intermediates starting with pyruvate. When you degrade pyruvate, you capture some of the energy in carbon carbon bond which is a high energy bond. this acetyl-CoA bond was kind of like the high energy body of ATP Alpha ketoglutarate is an intermediate for glutamate.
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Figure 10.16- Total yield of ATP during aerobic respiration. Assumes a P/O ratio of 2.5 for NADH oxidation and 1.5 for FADH2 oxidation. P= 1 ATP = 4 H+
O = 1/2O2 = 2e- =1 NADH = 10 H+
for FADH2, 2e- = 6 H+ Prokaryotes often have a lower yield because their electron transport chains translocate fewer H+ per 2e- and the PMF is used for more than ATP biosynthesis.
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Figure 10.19- Fermentation, an alternative to aerobic respiration. In the absence of an external electron acceptor, cells reduce pyruvate produced from glycolysis to regenerate NAD+ for glucose oxidation. A wide variety of different pathways are found in different prokaryotes. Growth is then possible from the ATP generated by substrate level phosphorylation. How do these cells make their PMF?
Suraj
Note
in fermentation, use glycoslyis to generate pyruvate and then use pyruvate as a sink to oxidize NADH. You can either reduce the pyruvate to lactate. Another alternative is to decarboxylate pyruvate. Another is to make acetyl CoA and make ethanol from there. Major purpose of these pathways is to get rid of the NADH (oxidize NADH). The reason you want to do that is you have to have enough NAD to oxidize glucose. If you run out of NAD, cells cannot oxidize glucose and cannot make ATP by substrate level phosphorylation. This pathway is also used for beta-hydroxybutarate biosynthesis. This series of reactions starting from pyruvate to acetylCoA and then on is very important and this is one way cells can make ATP via substrate level phosphorylation.
Suraj
Note
A lot of anaerobes are mobile and we know they have flagella and how does a fermentative organism use the PMF? --> All enzymes are reversible so you can make ATP in an aerobe by using ATP synthase. In an anaerobe, we use that enzyme to make PMF. Run ATP in reverse. It will make ATP using substrate level phosphorylation How does an aerobe make ATP? Using ATP synthase and make PMF
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Simple sugars and other monomers are rare in nature. Most of the carbon is in polymers such as cellulose, hemicellulose, chitin, lignin, proteins, fatty acids, etc. How do cells get this material? 1. Production of extracellular enzymes to degrade polymers too large to be
taken up by cells, such as proteases 2. Attachment to polymers and production of cell-associated degradative
complexes, such as the cellulosome
Roy H. Doi & Akihiko Kosugi Nature Reviews Microbiology 2,
541-551 (July 2004)
Suraj
Note
How do cells take up these material? --> For polymers, they will attach the polymers and will produce cell associated degradative; complexes. Example is the cellulosome. These particular bacteria have an S layer, which is a monolayer of proteins on the surface of the cell. They have a special protein that anchors into the S layer and prdouces this scaffold protein on the outside of the cell. Attached to this are enzymes that are glycolytic and are capable of degrading the cellulose substrate. Why do this? 1) You can keep the concentration of enzymes really high. The consequence is that when you produce the monomers or smaller pieces of sugards, they are right next to the cell and they are taken up. This is a way for a cell which cannot take up large particles can get this material
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Figure 10.20-Uptake of other sugars. Hexoses are converted to intermediates of glycolytic pathways, such as glucose-6-P. Disaccharides are converted to monosaccharides. Reserve polymers, such as glycogen and starch, also cleaved by phosphorylases:
(glucose)n + Pi (glucose)n-1 + glucose-1-P
Suraj
Note
Once you produce the monomes, you still need to plan galactose to glucose is an isomerization. Cellulobiose is a dimer formed from A
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Figure 10.21-22-Lipid catabolism Triglycerides hydrolyzed to glycerol and fatty acids by lipases, glycerol then degraded via the glycolytic pathway, and fatty acids often oxidized via β-oxidation pathway. The reserve material, poly(b-OH butyrate) also metabolized in this fashion.
Suraj
Note
This is similar and you are now hydrolyzing the glycerol which is degraded by glucose. The purpose here is to produce acetyl-CoA for TCA cycle. What do these reactions look like? TCA cycle. You can start with succinate and oxidize to fumarate, add water to malate, etc. This works for fatty acids; but it also works for polybetahydroxybutarate! Beta oxidation means oxidation is occuring at the Beta carbon. Alpha carbon is carbon with the COOH group (carbonyl).
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Figure 10.23-Protein and amino acid catabolism 1. Proteases hydrolyze proteins to peptides and amino acids, which are
taken up by the cell 2. Amino group is removed from the amino acid by deaminations or
tranaminations. 3. Resulting organic acids are converted to pyruvate, acetyl-CoA, or TCA
cycle intermediate 4. Oxidized by TCA cycle
What is the other keto acid in the TCA cycle? What amino acid does it form?
Suraj
Note
alpha ketoglutatarate is an intermediate in the TCA cylce and it forms glutamate
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Aromatic degradation Lignin is one of the most abundant organic compound on earth. Produced by woody plants, it is very recalcitrant and accumulates in soil. Many other aromatic compounds are important xenobiotics and pollutants.
Suraj
Note
Xenobiotics compounds are degraded by bacteria and are made my man SOM = soluble organic matter Know the basic definitions of Table 29.1 These are empirical definitions and don't refer to a specific compound
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Aromatic degradation Degradation proceeds stepwise: 1. Polymers converted to
monomers 2. In aerobes, monomers
converted to catechol or protocatechuate and then to TCA cycle intermediates by the b–ketoadipate pathway
3. In anaerobes, monomers are degraded to benzoate and acetyl-CoA
Suraj
Note
What amino acids might this be made from? --> Aromatic amino acids include tyrosine, phenylalanine, and tryptophan. Tyrosine and alanine are the major precursors for lignin biosynthesis.
