SITE IS DOWN FOR THE SUMMER

Older blogs below

Photosynthesis

This covers most of the chapter except for the alternative pathways up to 199, which is the beginning of the section on C4 and CAM plants.
Remember to read the Entry in LabArchives about the photosynthesis lab.

Photosynthesis:


Plants are examples of “Autotrophs,” or, organisms that can support themselves and don’t need to eat other things (we are “heterotrophs;” we do eat other things). In particular, plants are “photoautotrophs,” supporting themselves with light.
484px-Simple_photosynthesis_overview
This is the basic scheme we have been following. Photosynthesis can be broken into two parts, the “photo” part (light reactions” and the “Synthesis part” (Calvin Cycle). The light reactions take place in the thylakoid membrane and produce two main things: ATP and NADPH (It’s just like NADH, except it has an extra phosphate on the ribose…at the 2' position, if you care). The processes that result look like the ones we learned about in respiration (except, backward). The ATP synthase works the same way as it does in respiration.
The ATP and reduced nucleotide cofactor (NADPH) are used in the synthesis part (Calvin Cycle) to pull CO
2 out of the air and synthesize carbohydrates. In the process, the oxidized NADP+ and ADP are produced…and fed back into the light reactions.

Light Reactions


The general idea is that photons of light hit electrons, exciting them to a higher energy level. Instead of “falling” back down and emitting light, the high energy electrons are passed to other molecules, reducing them, and the electrons are passed down an electron transport chain of proteins or smaller molecules that are reduced, then oxidized by the next player in the chain. The actual electron transport system is simpler in the chloroplast than in the mitochondrion. However, it includes two separate places where electrons are excited by light.
Just like in respiration, electron transport moves protons across the membrane and those drive the ATP synthase. The first recipient in the chain is called “Plastoquinone” and it looks like this:
Plastoquinone
(That structure in the brackets is repeated 9 times). (The protein complex that gets the electrons next is called “cytochrome b6f”).You don’t have to know what all of them are.
There are two different photosystems (called
PSI and PSII) that can work together. PSI can also work on its own. Each has their own chlorophyl. In a pattern known as the “Z scheme,” electrons starting in PSII get passed though electron transport to PSI, where light excites it. Another electron RedOx reaction can pass two electrons onto NADP+, generating NADPH, the reduced form. This will be passed to the “synthesis” part, or Calvin Cycle. The Z scheme looks like this:Thylakoid_membrane

Some key points:
  1. In the Z scheme, PSII is excited “first,” (there are many reaction centers all being hit more or less simultaneously. It’s just that to see the “scheme,” we have to imagine things happening in sequence).
  2. Electrons leave PSII, passed to Plastoquinone (PQ). They are replaced by electrons taken from oxygen in water (a rare case of oxygen being oxidized). This generates O2, which is good for us.
  3. At some point PSI also gets hit by a photon of the right energy. That electron is can be passed directly to an iron-containing protein called “Ferrodoxin” and eventually used to reduce NADP. The electrons are replaced by electrons coming down the from the PSII system.
  4. The Z scheme is also called “linear flow” of electrons because they appear to start at one place (PSII) and pass down the chain, eventually leaving the system.
  5. Photosystem 1 (PSI) can work on its own without PSII. In that case the electrons excited from PSI get passed down the chain to Cytochrome b6f and passed back into a PSII reaction center. When this happens: Oxygen is NOT released and NADPH is not made. In some organisms, the electrons can be taken from some other source and NADPH can be made, but, oxygen is not produced.
  6. From an evolutionary point of view, PSI appears to be older. If that’s the case, photosynthesis was going on for a long time without oxygen being produced.

I’ve decided to do this in two parts…a somewhat watered down version, similar to what the book does and then a separate part that gets into more detail. You can read the detail or not. It’s up to you. You won’t be tested on it.

PhotoSYNTHESISBasic. There are three phases to the synthesis part.


  1. Carbon Fixation: The starting molecule is Ribulose 1-5 bisphosphate, a 5-carbon sugar derivative with a phosphate on each end. CO2 is pulled from the air and attached to one end, creating a very unstable 6-carbon molecule that breaks almost immediately in to two 3-carbon molecules called 3 phosphogycerate. If you’re counting Carbons, you started with 5, added 1 CO2 to get 6, then broke that into two 3-carbon molecules.
  2. Reduction: This molecule may be used for some biosynthetic pathways. However, to be really useful, it needs to be reduced from the carboxyl (COO-) form to the aldehyde form (C=O). This goes in two steps involving first ATP, then reduction with NADPH. This yields the very useful glyceraldehyde 3-phosphate (G3P). Do you remember that one from glycolysis?
  3. Regeneration: For it to be a “cycle,” the starting material Ribulose, 1-5 bisphosphate has to be regenerated. This is actually has several steps and if you don’t look a little at the details, the math doesn’t seem to work. For that reason, I’ll do a little detail here. What happens is the first two steps are run 3 times, (requiring 3 Ribulose 1-5 bisphosphate molecules), which gets you 6 G3P. One of those can be syphoned off to use for other things such as glucose synthesis. The remaining 5 G3P are rearranged through several enzymatic steps to get you back your 3 Ribulose 1-5 bisphosphate. (Five 3-carbon molecules get you three 5-carbon molecules…15 carbons in each case)
So, running the Calvin cycle 3 times gets you a net gain of one G3P. Run it 3 more times and you can use two of those to make a glucose. That takes more ATP and is essentially the reverse of the first few steps of glycolysis. It takes a total of 6 times round the cycle to make a single glucose.



Detail

Below I describe the process in more detail. You would never be held accountable for this. But, I thought some of you might like to read it.
  • We start with Ribulose 1-5 bisphosphate (that means 2 phosphates, one on each end of the molecule at positions 1 and 5). Note that ribulose is a ketose, with a carbonyl at position 2. I believe the attack of the CO2 is at position 4, one from the right end.
  • RuBP-2D-skeletal
  • The intermediate, 3-keto-2-carboxyarabinitol-1,5-bisphosphate, is immediately broken into two 3-phosphoglycerates. These have a phosphate on position 3, a carboxyl group (acid) at position 1. Note that here they are assumed to be deprotonated, as they would be at normal pH.rubismech
  • Phosphoglycerate can itself be used as starting material for many biosynthetic pathways. Some molecules may get syphoned off for other uses.
  • The enzyme phosphoglycerate kinase (which you may remember from glycolysis) uses ATP to add a phosphate to the other end (carbon 1) of 3-phosphoglycerate, yielding 1,3 bisphosphoglycerate (you need two ATP because you have two 3-PG). Note that the carboxyl end (left side) now goes COOPO3-. In ATP hydrolysis, ADP would be left with the extra oxygen and the Pi picks up its fourth O from water. Here, the transferred Phosphate gets its fourth Oxygen from the carboxyl.
    BIPGLYC
  • Recall that NADPH is a reducing agent...that is, it is oxidized easily. Note that if you now remove the Pi, the oxygen that was the end of the carboxyl is removed, leaving just the carbonyl. So, it’s now an aldehyde (terminal carbonyl) and is Glyceraldehyde 3-phosphate. (you know, G3P from the song). It can be used in biosynthetic pathways too. The H from NADPH is now at the end of the molecule, where the O used to be. This is carried out by an enzyme just like the one we see in glycolysis, Glyceraldehyde phosphate dehydrogenenase…just in the opposite direction.
  • Once you build up enough G3P, you can syphon one off for Glucose synthesis (note that this particular version of the cycle implies that it’s the 3 phosphoglycerate that is syphoned off. This is a bit of a controversy...and may just be a point of view. Let’s stick with the G3P as being the main intermediate used to build glucose.
  • I’m not going to go into any more detail on the regeneration process than I did above in phase 3. Just know that it is a multi-step pathway.



More study hints

I may update this later. But here are some more points to look into. Read More...

Study hints

I will try to update this with more stuff later. Read More...

Membrane Transport

This is a blog that covers earlier stuff…but might be a useful review.
The job of the membrane is to keep the “outside out” and the “inside in.” However, it is critical that some molecules get across the membrane. Very few things can just diffuse across the membrane (most things that would be soluble in the aqueous environment wouldn’t pass through the lipids). Thus, the job of transporting things across the membrane falls largely to proteins, and it is tightly regulated. We will go over some of the high points.
Read More...

Glycolysis

  • Glycolysis:
  • Here is the overall scheme:
Glycolysis2.svg
I will outline all the steps below.
The key points are:
  • Takes place in the cytoplasm
  • Separate into “investment phase” and “payoff phase”
  • Input is glucose or similar simple sugar
  • Output molecules are: Pyruvate, which can go to the mitochondria for further processing; NADH, which can be used in other reactions including to feed electrons into oxidative phosphorylation; 4 ATP…but only 2 net gain
  • Has an important regulated enzyme: phosphofructokinase (PFK). This enzyme is target for feedback to either increase or decrease glycolysis.
  • Investment phase results in fructose (6-carbon sugar) with a phosphate on both the first and sixth carbon (Fructose 1,6 bisphophate).
  • Payoff phase occurs after this is broken into two 3-carbon sugars, each with a phosphate on them. The most important one of these is glyceraldehyde-3 phosphate (G3P)

Feedback:


The idea of feedback was addressed in the Enzyme chapter and it is an important concept. This is just one really interesting example we will do in more detail.
If there is an excess of ATP available in the cell and the citric acid cycle is running at full capacity, ATP will bind to an
allosteric site on PFK (allosteric means it binds someplace else on the protein other than the active site…this would be a non-competitive inhibitor). ATP is also a substrate for PFK. This is not a contradiction because there are two different sites. The active site, where phosphate is transferred to the fructose 6-phosphate, binds with high affinity (That means it binds even if there is a low concentration of ATP). The separate, allosteric site binds with low affinity. So, it is only occupied if there is high concentration of ATP around. Binding of ATP to the allosteric site inhibits the enzyme.
There is also an inhibitory site for citrate.

Gluconeogenesis (this is not in the book, but is on the scary chart)


Here’s a challenging idea for you:
Like all enzyme pathways, glycolysis can be run backwards or forwards. In your liver “gluconeogenesis” (literally: making new glucose), can use
all but one of the enzymes of glycolysis to form glucose from oxaloacetate “siphoned” off the citric acid cycle, if energy production needs to be reduced. The role of that enzyme has to be carried out by a different protein. What do you think is that one enzyme is? It’s phosphofructokinase.
It’s not that the enzyme cannot work in reverse. Have any idea why PFK cannot be used, practically, in gluconeogenesis?
Here’s a hint: Gluconeogenesis would only occur if the cell had excess ATP.

Glycolysis,

all the steps (you are not supposed to memorize these. The important stuff is in bold):
  • Hexose Kinase (1st step), product is glucose 6-phosphate, keeps glucose inside cell and keeps gradient “downhill” into the cell. This uses an ATP, but is not regulated. This is the first “investment.”
  • 2nd step rearranges it to fructose 6-phosphate. That is, the hydroxyl of position 2 oxidizes the carbonyl on position 1. Position 1 gets the H to make a hydroxyl there and position 2 becomes a carbonyl. The enzyme can be called “hexose-phosphate isomerase” or, as it is on the diagram, phosphoglucose isomerase. Like most steps, this is freely reversible.
  • This is the rate-limiting and tightly regulated step. Phosphofructokinase (3rd step). This adds a second phosphate to fructose 6-phosphate, making fructose 1,6-bisphosphate. The regulation is via an allosteric site that binds ATP. Now, you have to know two things here: the enzyme has 2 sites that bind ATP, one in the active where it is necessary as a substrate for the reaction and a second that inhibits the reaction; the first site binds with higher affinity than the second. So, if the concentration of ATP is low to average in the cytoplasm, only the active site binds ATP. However, if the concentration of ATP gets high, indicating that more ATP is really not needed, the second, inhibitory site becomes occupied and shuts the enzyme off.
  • Aldolase (4, clips the six-carbon sugar diphosphate into 2 Triose phosphates called dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (G3P). G3P is the more useful one and the two are freely interconverted by triode isomerase. Since there are two, G3P made, all the subsequent steps can be done twice for each glucose.
  • The next step is carried out by Triose Phosphate Dehydrogenase (takes hydrogen off a triose phosphate). If something is “dehydrogenated” it is the same as being oxidized. Note, it’s not taking off an H+, it is an H with its electron. The thing that is reduced is NAD+. If you are interested in the chemistry here, it’s kind of interesting. Attacks position 1, the carbonyl. The exchange results a carboxyl with a phosphate on it. That’s a really high-energy phosphate. Hydrogen from the carbonyl carbon is transferred to NAD+, reducing it. This is the first energy payoff in that it creates two NADH + H+ (one for each of the G3P), which can be used in oxidative phosphorylation or as a high-energy cofactor in certain other reactions.
  • The first "Payoff." Substrate-level phosphorylation. The phosphate on position 1 doesn’t stay long, as it is such a high-energy phosphate. It is transferred to ADP to make ATP in the next step. This process is called “substrate-level phosphorylation,” and is carried out in this case by the enzyme phosphoglycerate kinase. I know what you are thinking: “Wait…shouldn’t that name mean it adds a phosphate to 3-phosphoglycerate?” How clever of you to notice. The enzyme is named for the reverse reaction to the one we are talking about as part of glycolysis. There are several key enzymes for which this is the case. Important concept: It turns out that if you purify it away from everything else, it seems to favor the reverse reaction. It can drive it either way. The reason the reaction goes primarily toward producing phosphoglycerate is because there IS a next step. You see, as soon as 3-phosphoglycerate is formed, it gets pulled into that step.
  • Next is a simple rearrangement where the phosphate gets moved from position 3 to position 2.
  • 2-phosphoglycerate then undergoes a step carried out by a fairly famous enzyme: Enolase. This enzyme pulls a water molecule out but makes the most unstable, highest energy molecule in the series, Phosphoenolpyruvate (PEP). Enolase is essential in every cell that does glycolysis. It is rather famously inhibited by fluoride ions. This is why we put fluoride in toothpaste (and, in less conspiracy-minded communities, in the water). The fluoride kills the bacteria in your mouth. Anyway, that phosphate next to the double bond has to go.
  • Second Payoff Step. The last step takes that phosphate and transfers it onto ADP to make one more ATP. The product is pyruvate, which goes off to the mitochondria for the TCA cycle. Again, this enzyme has a backwards name.

Here is a link to a
glycolysis rap…it’s accurate and not too painful to watch…just a little creepy.

Fermentation

Fermentation:


As usual, I’ve taken all these images from Wikipedia…because I can.
We have been discussing how aerobic (in the presence of O2) respiration works. Importantly, there are some odd organism that use other electron acceptors when there is no oxygen. There are some iron and sulfur compounds used by some odd bacteria in anaerobic (no oxygen) conditions. Their electron transport systems are a little different, but the concept is the same. All eukaryotes use Oxygen as the electron acceptor if they use mitochondria at all. However, Archaea and Bacteria are far more versatile.

The problem: running out of NAD+


If Eukaryotes have no oxygen, then what? Well, we can still run glycolysis to obtain ATP by breaking down glucose and other molecules. It’s not very efficient and generates only small amounts of ATP. But, there is a bigger problem: what to do with the NADH?
Glycolysis uses NAD
+ as an oxidizing agent, generating the reduced form: NADH + H+. That’s a good thing if there is oxygen around because we transport that to the mitochondria and use it to feed electrons into the ETS (which oxidizes it and generates the NAD+ again). If there is no oxygen, we run out of NAD+ and we cannot even continue glycolysis. We also have a build up of pyruvate we really cannot use.
We “solve” these problems by reducing pyruvate to lactic acid, using NADH as the reducing agent. That regenerates the NAD
+.
This is pyruvic acid, (since the proton is still attached to the carboxyl in this diagram, it’s named as the acid):
Pyruvic-acid-3D-balls. “Pyruvate” is the name for the form that has lost it’s H+, also known as the conjugate base. It looks like this, with a negative charge on the oxygens on the right:751px-Pyruvate-3D-balls
this is Lactic acid
: 753px-Lactic-acid-3D-balls. Notice that the middle carbon was a carbonyl, and now is a hydroxyl. That’s where the reduction is taking place.
Unfortunately for you, the lactate really makes your muscles hurt and not work so well.
Another organism that does this form of fermentation are “Lactobacilli.” These are the strains of bacteria that make yogurt.

Beer, Bread and Wine:


But, there is still another form of fermentation that does one additional step. While there are other organisms that do this form of fermentation, by far the most successful are strains of yeast. They are successful because what they end up making is very popular among humans. The first step in this fermentation is to remove that carboxyl group from the end of pyruvate. This releases CO2 as a gas. You are then left with a toxic and bad-smelling molecule called acetaldehyde: 624px-Acetaldehyde-3D-balls
This molecule is then reduced, to ethanol (as before, the carbonyl is converted to a hydroxyl.
Ethanol-3D-balls and NADH is oxidized back to NAD+. The widespread use of yeast to carry out this reaction on sugars from various grains or fruit has given us bread (the CO2 is what causes bread to rise), beer and wine (the alcohol is very popular in some circles). As a side benefit, the yeast is able to make a wide range of nutrients that humans need, notably B vitamins. Bread made this way has a lot more nutritive value than the starting flour. Beer also has a lot of nutrients.
Although it does not portray the mitochondrial
outer membrane, this is a good general diagram of what we have been studying:

Cellular_respiration

A couple of names: “Proton motive force” is a name given to the electrochemical gradient of protons that drives the ATP synthase. Similarly “chemiosmosis” is another name for the protons driving the ATP synthase. Electron Transport and Chemiosmosis together comprise "Oxidative phosphorylation."

Enzyme Cofactors

Many enzymes require “cofactors.” These are really second substrates that are used for the chemical reaction. You will find many of them are molecules that fall into the “Vitamin” category. That is, many vitamins are molecules used by enzymes to carry out chemical reactions. Read More...

Respiration 1

Cellular Respiration.

Here is a link to a screencast from last year.

We break down glucose (and lots of other things) to make CO
2 and H2O. Both are much lower in chemical potential energy than the starting food item, so free energy is released. But, the convoluted steps we use to do this allow us to harvest little bits of energy to make molecules with a convenient amount of chemical potential energy (such as ATP and NADH+H+).
Big Picture:
Remember that oxidation/reduction (RedOx) reactions involve electrons being traded from positions of high potential energy to ones of lower potential energy, with the resulting release of some free energy (the difference, or delta) between the start and final state.
The overall reaction in respiration combines carbon and hydrogen gotten from carbohydrates and other carbon-containing molecules with oxygen to make CO
2 and water. But, instead of doing that all at once, there is a long series of reactions releasing smaller amounts of free energy, some of which is captured in nice, “bite-sized,” chunks of energy we can use. What “bite-sized” chunk really means is electrons in compounds easily oxidized. The two main bites of energy for the cell are ATP and NADH.
We will produce ATP through simple reactions called “substrate level phosphorylation” and through oxidative phosphorylation. In this second process, NADH is the source of electrons for the electron transport system, which uses RedOx reactions to pump protons to the inter-membrane space. Finally, the ATP synthase uses that proton gradient to synthesize ATP.
We will start at the end, because it makes more sense this way.

ATP Synthase


  • Enter the ATP synthase:
  • Atp_synthase
    This machine is almost too good to be true. It’s in the mitochondrial inner membrane, the chloroplast inner membrane and the plasma membrane or inner membrane of bacteria. The protons flow down gradient through a channel, which leads to mechanical changes that spin the rotor (more like a ratchet). This mechanical energy is then used to drive ATP synthesis. Thus, we convert mechanical energy to chemical potential energy. The process is also called “chemiosmosis.”
  • Too cool. Here is a video. It’s not the one from the creationist site.

All this needs to run is a proton gradient. How do you get that? Well, that depends. In the mitochondria and chloroplast, the proton gradient comes from a system called “Electron Transport,” also taking place in the inner membrane of the mitochondria (or chloroplast).

Reducing equivalents or “Reduced Cofactors”


To run electron transport, you need electrons in a high energy state. That is, you need something that has electrons that are relatively easy to remove to a lower state. The thing donating the electrons is the “reducing agent” (it is oxidized) and therefore we call it a “reducing equivalent” or “reduced cofactor.” It will be one or more “B” vitamin derivatives in its reduced form.

  • A word about “cofactors.” I hope all of you were starting to see the patterns in the nucleotide cofactors. Take a look at the separate entry on cofactors.
  • NAD clearly has a lot in common with ADP. NADH is sometimes used directly in enzymatic reactions requiring energy. These really are variations on a theme. The cofactors in electron transport are variations on another theme, or, pardon the pun, variations on a Heme. These are the iron-containing rings that you are familiar with from Hemoglobin, which can carry oxygen in your blood. But, similar cofactors will be used in electron transport.
  • If you want to carry electrons in a membrane, you need something that is hydrophobic to do it. The go-to cofactor there is “Coenzyme Q.” It is not based ADP. The “MeO” in the structure stands for a Methyl-Oxygen or “methyl ether.”
CoQ
  • You will see these structures over and over. They will always be doing similar tasks.

Oxidative phosphorylation.

  • Technically, this includes two stages: the phosphorylation by the ATP synthase we’ve already discussed, and the Electron Transport System which uses oxidation/reduction reactions to pump protons to the inter-membrane space to drive the ATP synthase.
  • I stole the figure below from a great text book called “Molecular Cell Biology.”

ETS2
The key players here are called either electron carriers such as Cytochromes and cofactors such as coenzyme Q, or oxido/reductases, enzymes that catalyze the transfer of electrons, frequently while pumping protons across the membrane. You don’t need to know the names of them, though I might use them. The complexes are just labeled with Roman Numerals.
The cytochromes are small-ish proteins that contain bound within them, usually, an Iron atom/ion. This is cytochrome c, one of the
  • Most well characterized cytochromes.Cytochrome_C The multi-ring structure in the middle is not part of the protein per se. It is called a “heme,’ as in “hemoglobin,” and is held in place by the protein. The red dot is an iron. Look at the general scheme on page 171, figure 9.13. Electrons are passed from NADH or FADH2. These enter the “electron transport system” which is the string of cytochromes and other acceptor/donor molecules in the inner membrane. The electrons pass down through a series of RedOx reactions, the acceptor for one step being the donor for the next. Finally, the electrons are passed to Oxygen, which pairs with protons to make water.
  • At three steps along the way, just due to the orientation in the membrane, the H+ product of the dehydrongenation (oxidation) is spit into the intermembrane space (between the two membranes—we’re so clever with names). This leads to an electrochemical gradient.
  • Key Points:

  1. The donor of electrons are cofactors NADH+ and FADH2 .
  2. The oxidation/reduction reactions pass electrons from one carrier to another in or near the membrane.
  3. The reactions result in protons being pumped to the inter-membrane space, establishing the electrochemical gradient that runs the ATP Synthase.
  4. The final recipient of the electrons is oxygen, which combines with H+ to make water. That’s why you breath out water.
The remaining question is: Where did the reduced cofactors NADH and FADH2 come from? We’ll get to that next.

Enzyme Lecture

Enzyme Lecture:
This is a long lecture (close to an hour) that has a lot in it. Could be useful.
It covers most of chapter 8.

Enzyme Kinetics


Read More...