Second Messengers

Second messengers are molecules that relay signals received at receptors on the cell surface — such as the arrival of protein hormones, growth factors, etc. — to target molecules in the cytosol and/or nucleus.
But in addition to their job as relay molecules, second messengers serve to greatly amplify the strength of the signal. Binding of a ligand to a single receptor at the cell surface may end up causing massive changes in the biochemical activities within the cell.
There are 3 major classes of second messengers:

1. cyclic nucleotides (e.g., cAMP and cGMP)
2. inositol trisphosphate (IP₃) and diacylglycerol (DAG)
3. calcium ions (Ca²⁺)

Cyclic Nucleotides

Cyclic AMP (cAMP)

Some of the hormones that achieve their effects through cAMP as a second messenger:

• adrenaline
• glucagon
• luteinizing hormone (LH)

Cyclic AMP is synthesized from ATP by the action of the enzyme adenylyl cyclase.

• Binding of the hormone to its receptor activates
• a G protein which, in turn, activates
• adenylyl cyclase.
• The resulting rise in cAMP turns on the appropriate response in the cell by either (or both):
  • changing the molecular activities in the cytosol, often using Protein Kinase A (PKA) — a cAMP-dependent protein kinase that phosphorylates target proteins;
  • turning on a new pattern of gene transcription. [View mechanism]

Cyclic GMP (cGMP)

Cyclic GMP is synthesized from the nucleotide GTP using the enzyme guanylyl cyclase.
Cyclic GMP serves as the second messenger for

• atrial natriuretic peptide (ANP)
• nitric oxide (NO)
• the response of the rods of the retina to light. [Discussion]

Some of the effects of cGMP are mediated through Protein Kinase G (PKG) — a cGMP-dependent protein kinase that phosphorylates target proteins in the cell.

Inositol trisphosphate (IP₃) and diacylglycerol (DAG)

Peptide and protein hormones like

• vasopressin,
• thyroid-stimulating hormone (TSH), and
• angiotensin

and neurotransmitters like GABA
bind to G protein-coupled receptors (GPCRs) that activate the intracellular enzyme phospholipase C (PLC).
As its name suggests, it hydrolyzes phospholipids — specifically phosphatidylinositol-4,5-bisphosphate (PIP₂) which is found in the inner layer of the plasma membrane. Hydrolysis of PIP₂ yields two products:

• diacylglycerol (DAG)DAG remains in the inner layer of the plasma membrane. It recruits Protein Kinase C (PKC) — a calcium-dependent kinase that phosphorylates many other proteins that bring about the changes in the cell.
  As its name suggests, activation of PKC requires calcium ions. These are made available by the action of the other second messenger — IP₃.
• inositol-1,4,5-trisphosphate (IP₃)
  • This soluble molecule diffuses through the cytosol and
  • binds to receptors on the endoplasmic reticulum causing
  • the release of calcium ions (Ca²⁺) into the cytosol.
  • The rise in intracellular calcium triggers the response.Example: the calcium rise is needed for NF-AT (the "nuclear factor of activated T cells") to turn on the appropriate genes in the nucleus. [Discussion]
    The remarkable ability of tacrolimus and cyclosporine to prevent graft rejection is due to their blocking this pathway.

The binding of an antigen to its receptor on a B cell (the BCR) also generates the second messengers DAG and IP₃.

Calcium ions (Ca²⁺)

As the functions of IP₃ and DAG indicate, calcium ions are also important intracellular messengers. In fact, calcium ions are probably the most widely used intracellular messengers.
In response to many different signals, a rise in the concentration of Ca²⁺ in the cytosol triggers many types of events such as

• muscle contraction;
• exocytosis, e.g.,
  • release of neurotransmitters at synapses (and essential for the long-term synaptic changes that produce Long-Term Potentiation (LTP) and Long-Term Depression (LTD);
  • secretion of hormones like insulin
• activation of T cells and B cells when they bind antigen with their antigen receptors (TCRs and BCRs respectively)
• adhesion of cells to the extracellular matrix (ECM)
• apoptosis
• a variety of biochemical changes mediated by Protein Kinase C (PKC).

Normally, the level of calcium in the cell is very low (~100 nM). There are two main depots of Ca²⁺ for the cell:

• The extracellular fluid (ECF — made from blood), where the concentration is ~ 2 mM or 20,000 times higher than in the cytosol;
• the endoplasmic reticulum ("sarcoplasmic" reticulum in skeletal muscle).

However, its level in the cell can rise dramatically

• when channels in the plasma membrane open to allow it in from the extracellular fluid or
• from depots within the cell such as the endoplasmic reticulum and mitochondria.

Getting Ca²⁺ into (and out of) the cytosol

• Voltage-gated channels
  • open in response to a change in membrane potential, e.g. the depolarization of an action potential;
  • are found in excitable cells:
    • skeletal muscle
    • smooth muscle (These are the channels blocked by drugs, such as felodipine [Plendil®], used to treat high blood pressure. The influx of Ca²⁺ contracts the smooth muscle walls of the arterioles, raising blood pressure. The drugs block this.)
    • neurons. When the action potential reaches the presynaptic terminal, the influx of Ca²⁺ triggers the release of the neurotransmitter.
    • the taste cells that respond to salt [Link].
  • allow some 10⁶ ions to flow in each second following the steep concentration gradient.
• Receptor-operated channels
  These are found in the post-synaptic membrane and open when they bind the neurotransmitter. Example: NMDA receptors.
• G-protein-coupled receptors (GPCRs). These are not channels but they trigger a release of Ca²⁺ from the endoplasmic reticulum as described above. They are activated by various hormones and neurotransmitters (as well as bitter substances on taste cells in the tongue — Link).

Ca²⁺ ions are returned

• to the ECF by active transport using
  • an ATP-driven pump called a Ca²⁺ ATPase;
  • two Na⁺/Ca²⁺ exchangers. These antiport pumps harness the energy of
    • 3 Na⁺ ions flowing DOWN their concentration gradient to pump one Ca²⁺ against its gradient and
    • 4 Na⁺ ions flowing down to pump 1 Ca²⁺ and 1 K⁺ ion up their concentration gradients.
• to the endoplasmic (and sarcoplasmic) reticulum using another Ca²⁺ ATPase [Link].

How can such a simple ion like Ca²⁺ regulate so many different processes? Some factors at work:

• localization within the cell (e.g., released at one spot — the T-system is an example — or spread throughout the cell)
• by the amount released (amplitude modulation, "AM")
• by releasing it in pulses of different frequencies (frequency modulation, "FM")

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Serine Proteases

The serine proteases are a family of enzymes that cut certain peptide bonds in other proteins.

This activity depends on a set of amino acid residues in the active site of the enzyme — one of which is always a serine (thus accounting for their name).

In mammals, serine proteases perform many important functions, especially in digestion, blood clotting, and the complement system.

Digestive Enzymes

Three protein-digesting enzymes secreted by the pancreas are serine proteases [Link]:

• chymotrypsin
• trypsin
• elastase

These three share closely-similar structures (tertiary as well as primary). In fact, their active serine residue is at the same position (Ser-195) in all three.

Despite their similarities, they have different substrate specificities; that is, they cleave different peptide bonds during protein digestion. [More]

Clotting Factors

Several activated clotting factors are serine proteases, including

• Factors 10 (X), 11 (XI), and 12 (XII)
• Thrombin
• Plasmin

Complement Factors

Several proteins involved in the complement cascade are serine proteases, including

• C1r and C1s
• the C3 convertases
  • C4b,2a
  • C3b,Bb

Serpins

Serpins are Serine Protease Inhibitors.

Here is a list of a few important serine proteases and the serpins that control them.

Serine Protease	Serpin
Chymotrypsin	alpha-1-antichymotrypsin
Complement factor C1s	C1 Inhibitor (C1INH)
Elastase (secreted by neutrophils)	alpha-1-antitrypsin
Clotting factor 10 (X)	antithrombin III
Thrombin	antithrombin III
Plasmin	alpha-2-antiplasmin
Trypsin	pancreatic trypsin inhibitor

How Serpins Work

The serpins inhibit the action of their respective serine protease by mimicking the three-dimensional structure of the normal substrate of the protease.

• The serine protease binds the serpin instead of its normal substrate. This alone would block any further activity by the protease. But the serpin has another trick to play.
• The protease makes a cut in the serpin leading to
  • the formation of a covalent bond linking the two molecules;
  • a massive allosteric change in the tertiary structure of the serpin;
  • which moves the attached protease to a site where it can be destroyed.

Importance of Serpins

Almost 20% of the proteins found in blood plasma are serpins.

Their abundance reflects their importance: putting a stop to proteolytic activity when the need for it is over.

This is especially important for the

• clotting and
• complement

systems where a tiny initial activating event leads to a rapidly amplifying cascade of activity.

Serpin Deficiencies

A number of inherited human diseases are caused by a deficiency of a particular serpin. The deficiency usually results from a mutation in the gene encoding the serpin.

Examples:

Alpha-1-antitrypsin deficiency

Alpha-1-antitrypsin inactivates the elastase secreted by neutrophils. When the lungs become inflamed, neutrophils secrete elastase as a defensive measure. However, it is important to inactivate this elastase as soon as its job is done. That is the function of alpha-1-antitrypsin.

(Its name, alpha-1-antitrypsin, suggests that it attacks the digestive enzyme, trypsin. In vitro, it does, but in the body, alpha-1-antitrypsin is found in the blood, not the intestine. Inactivation of trypsin in the intestine is the function of another serpin, pancreatic trypsin inhibitor.)

People with an inherited deficiency of alpha-1-antitrypsin (they are homozygous for a point mutation in its gene) are prone to emphysema. An effective treatment is on the horizon now that genetic engineering has produced goats that secrete human alpha-1-antitrypsin in their milk. [More]

Alpha-1-antitrypsin deficiency can also lead to liver damage. Alpha-1-antitrypsin is synthesized in the liver. However, some mutant versions of the molecule form insoluble aggregates within the liver cells. This mechanism is similar to that of the prion diseases where protein aggregates destroy neurons in the brain [Link]. A drug that enhances autophagy protects mice from the liver damage caused by aggregates of mutant alpha-1-antitrypsin.

C1INH deficiency

A deficiency of C1INH produces hereditary angioedema. Patients are at risk of occasional explosive triggering of the complement system. The massive release of anaphylatoxins (C3a, C5a) may cause dangerous swelling (edema) of the airways, as well as of the skin and intestine.

C1INH also inhibits kallikrein, the enzyme responsible for forming the potent vasodilator bradykinin. Hence, a deficiency of C1INH can trigger a violent inflammatory response by this mechanism as well. [More]

Antiplasmin deficiency

A deficiency in antiplasmin puts the person at risk of uncontrollable bleeding.

Antithrombin deficiency

A deficiency in antithrombin puts the person at risk of spontaneous blood clots, which can lead to a heart attack or stroke.

In January 2009, an advisory committee of the U.S. FDA decided that a recombinant human antithrombin (ATryn®) secreted into the milk of transgenic goats was safe for use in therapy.

The Evolution of the Serine Proteases

The close sequence similarity of the various mammalian serine proteases suggests that each is the product of a gene descended by repeated gene duplication from a single ancestral gene.

Other Serine Proteases

Serine proteases and molecules similar to them are found elsewhere in nature.

Subtilisin

Subtilisin is a serine protease secreted by the bacterium Bacillus subtilis. Although it has the same mechanism of action as the serine proteases of mammals, its primary structure and tertiary structure are entirely different.

Here, then, is an example at the molecular level of convergent evolution: two molecules acquiring the same function (analogous) but having evolved from different genes.

Acetylcholinesterase

This enzyme is built like and acts like the other serine proteases, but its substrate is the neurotransmitter acetylcholine, not a protein.

It is found at several types of synapses as well as at the neuromuscular junction — the specialized synapse that triggers the contraction of skeletal muscle.

The organophosphate compounds used as

• insecticides (e.g., parathion) and
• nerve gases (e.g. Sarin)

bind to the serine at the active site of acetylcholinesterase blocking its action.

Serpinlike Molecules

Angiotensinogen

This peptide is the precursor of angiotensin II — a major factor in maintaining blood pressure.

Link to discussion of angiotensinogen.

Chicken Ovalbumin

This is the major protein in the "white" of the egg (and a favorite antigen in immunological research).

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Reactive Oxygen Species (ROS)

Reactive oxygen species are

• molecules like hydrogen peroxide (#5)
• ions like the hypochlorite ion (#6)
• radicals like the hydroxyl radical (#3). It is the most reactive of them all; note how it differs from the hydroxyl ion (#4).
• the superoxide anion (#2) which is both ion and radical.

A radical (also called a "free radical") is a clusters of atoms one of which contains an unpaired electron (shown in red) in its outermost shell of electrons. This is an extremely unstable configuration, and radicals quickly react with other molecules or radicals to achieve the stable configuration of 4 pairs of electrons in their outermost shell (one pair for hydrogen).

Link to discussion of electron organization in atoms.

ROS Formation

Reactive oxygen species are formed by several different mechanisms:

• the interaction of ionizing radiation with biological molecules
• as an unavoidable byproduct of cellular respiration. Some electrons passing "down" the electron transport chain leak away from the main path (especially as they pass throughubiquinone) and go directly to reduce oxygen molecules to the superoxide anion (#2 above).
• synthesized by dedicated enzymes in phagocytic cells like neutrophils and macrophages
  • NADPH oxidase (in both type of phagocytes)
  • myeloperoxidase (in neutrophils only)

ROS Activity

Strong oxidants like the various ROS can damage other molecules and the cell structures of which they are a part.

Among the most important of these are the actions of free radicals on the fatty acid side chains of lipids in the various membranes of the cell, especially mitochondrial membranes (which are directly exposed to the superoxide anions produced during cellular respiration).
The figure shows one common series of reactions.

• A hydroxyl radical removes a hydrogen atom from one of the carbon atoms in the fatty acid chain (only a portion of which is shown) forming
• a molecule of water and leaving the carbon atom with an unpaired electron (in red); thus now a radical.
• Several possible fates await it.One of the most likely (and shown here) is to react with a molecule of oxygen (O₂) forming a peroxyl radical.
  This might then steal a hydrogen atom from a nearby side chain making it now a radical.
  One of the insidious things about free radicals is that in interacting with other molecules to gain a stable configuration of electrons, they convert that target molecule into a radical. So a chain reaction begins that will propagate until two radicals meet each other and each contributes its unpaired electron to form a covalent bond linking the two.
  Two common examples:The peroxyl radical may interact with:
  
  • another peroxyl radical on a nearby side chain crosslinking them with a covalent bond.
  • another nearby carbon-centered radical crosslinking them covalently.
  In both these latter cases, radical formation comes to an end but with the result that the fatty acid side chains of membrane lipids may have become so deformed as to damage the membrane.
  The lipofuscin so characteristic of aging cells may be formed by these mechanisms [Link].

Defenses Against ROS

Cells have a variety of defenses against the harmful effects of ROS. These include two enzymes:

• superoxide dismutase (SOD), which converts two superoxide anions into a molecule of hydrogen peroxide and one of oxygen, and
• catalase

as well as several small molecules that are antioxidants, such as

• alpha-tocopherol (vitamin E). This can break the covalent links that ROS have formed between fatty acid side chains in membrane lipids.
• uric acid. (Perhaps the long life span of some reptiles and birds is attributable to their high levels of uric acid.)
• vitamin C (in the right concentration)

Pharmacy shelves are filled with antioxidant preparations that people take in the hope of warding off the damaging effects (perhaps including aging) of ROS.

ROS are Essential

But it is important that the attempt to limit the production of ROS not succeed too well, because ROS have important functions to perform in the cell.
Examples:

• The cells of the thyroid gland must make hydrogen peroxide in order to attach iodine atoms to thyroglobulin in the synthesis of thyroxine.
• Macrophages and neutrophils must generate ROS in order to kill some types of bacteria that they engulf by phagocytosis.
  • Bacteria are engulfed into a phagosome.
  • This fuses with a lysosome.
  • Subunits of the enzyme NADPH oxidase assemble in the lysosome membrane forming the active enzyme.
  • It catalyzes the synthesis of the superoxide anion.
    NADPH − 2 e⁻ + 2O₂ −> NADP⁺ + H⁺ + 2 ^. O₂⁻
  • This activity produces a large increase in oxygen consumption, called the "respiratory burst".
  • Superoxide dismutase (SOD) converts this into hydrogen peroxide, which kills off the engulfed bacteria (except those that manufacture enough catalase to protect themselves).
• Neutrophils (but not macrophages) also kill off engulfed pathogens by using the enzyme myeloperoxidase which catalyzes the reaction of hydrogen peroxide (made from superoxide anions) with chloride ions to produce the strongly antiseptic hypochlorite ion (OCl⁻, #6 above).
  H₂O₂ + Cl⁻ −> HOCl (hypochlorous acid) + OH⁻
  HOCl −> H⁺ + OCl⁻

Chronic Granulomatous Disease (CGD)

This rare genetic disorder demonstrates the importance of ROS in protecting us from many type of bacterial infection. It is caused by a defective gene for one of the subunits of NADPH oxidase.
People with CGD have a difficult time ridding themselves of bacterial infections — especially those caused by bacteria (e.g. staphylococci, Salmonella) and fungi (e.g., Aspergillus) that produce catalase to protect themselves against the hydrogen peroxide generated by the macrophages and neutrophils that engulf them. Often the result is the development of a persisting nest of infected cells — called a granuloma.
The gene for one of the subunits of NADPH most frequently mutated in CGD is on the X chromosome. Thus males are principally affected [More].
However, examination of the neutrophils of females who are carriers of the gene shows that 50% of them do not make active NADPH oxidase when they engulf pathogens. In these cells, the X chromosome with the nonmutant allele has been inactivated and converted into a Barr body. [Link to discussion]
In June 2005, two cases of successful gene therapy for CGD were reported. Blood stem cells from the patients were removed, and the active gene for the NADPH subunit inserted into them using a retroviral vector. The transformed cells were returned to the patients, took up residence in their bone marrow, proliferated successfully, and improved their symptoms.

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The Energy Relationships in Cellular Respiration and Photosynthesis: the Balance Sheet

The respiration (or burning) of a mole of glucose releases 686 kcal of energy. This value represents the difference between the energy needed to break the bonds of the reactants (glucose and oxygen) and the energy liberated when the bonds of the products (H₂O and CO₂) form.

Average bond energies, kcal/mole
C-H	98
O-H	110
C-C	80
C-O	78
H-H	103
C-N	65
O=O	116 (2 x 58)
C=O	187* (2 x 93.5)
C=C	145 (2 x 72.5)
(* as found in CO2)

Conversely, the photosynthesis of a mole of glucose requires the input of 686 kcal of energy.
The reasons:

• water and carbon dioxide
  • the differences in electronegativity between their atoms are high
  • so they have polar covalent bonds with high bond energies
  • these are strong bonds
    • broken with difficulty and
    • liberating copious amounts of energy when they form
• glucose and oxygen
  • the differences in electronegativity between their atoms tend to be lower
  • so they form covalent bonds with average bond energies on the low side
  • these are broken with relative ease

The diagram shows the details.
 

The overall equation for each process is the same; only the direction of the arrow differs. (The actual equation is:
but we shall ignore the six molecules of water that occur on each side as they "cancel out".)
The structural formulas are shown as well as the average bond energies for each bond involved.

Cellular Respiration

As you can see,

• the 24 moles of covalent bonds in a mole of glucose require a total of 2182 kcal to be broken.
• The 6 double bonds of oxygen require another 696.

As for the products,

• The formation of 6 moles of CO₂ involves the formation of 12 double polar covalent bonds each with a bond energy of 187 kcal/mole; total = 2244
• The formation of 6 moles of H₂O involves the formation of 12 O-H bonds each with an energy of 110 kcal/mole; total = 1320.

The minus sign indicates that free energy has been removed from the system.

Link to discussion of free energy.

Photosynthesis

The details of the energy budget are just the same. The only difference is that now it takes 3564 kcal to break the bonds of the reactants and only 2878 kcal are released in forming glucose and oxygen. So we express this change in free energy (+686 kcal) with a plus sign to indicate that energy has been added to the system. The energy came from the sun and now is stored in the form of bond energy that can power the needs of all life.

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Photosynthesis: The Role of Light

The heart of photosynthesis as it occurs in most autotrophs consists of two key processes:

• the removal of hydrogen (H) atoms from water molecules
• the reduction of carbon dioxide (CO₂) by these hydrogen atoms to form organic molecules.

The second process involves a cyclic series of reactions named (after its discoverer) the Calvin Cycle. It is discussed in Photosynthesis: Pathway of Carbon Fixation. The details of the first process is our topic here.
A description of some of the experiments that led our understanding of these processes are described in Discovering the Secrets of Photosynthesis.
The electrons (e⁻) and protons (H⁺) that make up hydrogen atoms are stripped away separately from water molecules.

2H₂O -> 4e⁻ + 4H⁺ + O₂

The electrons serve two functions:

• They reduce NADP⁺ to NADPH for use in the Calvin Cycle.
• They set up an electrochemical charge that provides the energy for pumping protons from the stroma of the chloroplast into the interior of the thylakoid [View].

The protons also serve two functions:

• They participate in the reduction of NADP⁺ to NADPH.
• As they flow back out from the interior of the thylakoid (by facilitated diffusion), passing down their concentration gradient), the energy they give up is harnessed to the conversion of ADP to ATP.
• Because it is drive by light, this process is called photophosphorylation.

  ADP + P_i -> ATP

  The ATP provides the second essential ingredient for running the Calvin Cycle.

The removal of electrons from water molecules and their transfer to NADP⁺ requires energy. The electrons are moving from a redox potential of about +0.82 volt in water to −0.32 volt in NADPH. Thus enough energy must be available to move them against a total potential of 1.14 volts. Where does the needed energy come from? The answer: Light.

The Thylakoid Membrane

Chloroplasts contain a system of thylakoid membranes surrounded by a fluid stroma.

Link to page on chloroplast structure.

Six different complexes of integral membrane proteins are embedded in the thylakoid membrane. The exact structure of these complexes differs from group to group (e.g., plant vs. alga) and even within a group (e.g., illuminated in air or underwater). But, in general, one finds:

1. Photosystem I

The structure of photosystem I in a cyanobacterium ("blue-green alga") has been completely worked out. It probably closely resembles that of plants as well.
It is a homotrimer with each subunit in the trimer containing:

• 12 different protein molecules bound to
• 96 molecules of chlorophyll a
  • 2 molecules of the reaction center chlorophyll P₇₀₀
  • 4 accessory molecules closely associated with them
  • 90 molecules that serve as antenna pigments
• 22 carotenoid molecules
• 4 lipid molecules
• 3 clusters of Fe₄S₄
• 2 phylloquinones

View structures of chlorophyll a, chlorophyll b, and beta-carotene, a carotenoid.

2. Photosystem II

Photosystem II is also a complex of

• > 20 different protein molecules bound to
• 50 or more chlorophyll a molecules
  • 2 molecules of the reaction center chlorophyll P₆₈₀
  • 2 accessory molecules close to them
  • 2 molecules of pheophytin (chlorophyll without the Mg⁺⁺)
  • the remaining molecules of chlorophyll a serve as antenna pigments.
• some half dozen carotenoid molecules. These also serve as antenna pigments.
• 2 molecules of plastoquinone

3. & 4. Light-Harvesting Complexes (LHC)

• LHC-I associated with photosystem I
• LHC-II associated with photosystem II

These LHCs also act as antenna pigments harvesting light and passing its energy on to their respective photosystems.
The LHC-II of spinach is a homotrimer, with each monomer containing

• a single polypeptide
• 8 molecules of chlorophyll a
• 6 molecules of chlorophyll b
• 4 carotenoid molecules

5. Cytochromes b₆ and f

6. ATP synthase

How the System Works

• Light is absorbed by the antenna pigments of photosystems II and I.
• The absorbed energy is transferred to the reaction center chlorophylls, P₆₈₀ in photosystem II, P₇₀₀ in photosystem I.
• Absorption of 1 photon of light by Photosystem II removes 1 electron from P₆₈₀.
• With its resulting positive charge, P₆₈₀ is sufficiently electronegative that it can remove 1 electron from a molecule of water.
• When these steps have occurred 4 times, requiring 2 molecules of water, 1 molecule of oxygen and 4 protons (H⁺) are released
• The electrons are transferred (by way of plastoquinone — PQ in the figure) to the cytochrome b₆/f complex where they provide the energy for chemiosmosis.
• Activation of P₇₀₀ in photosystem I enables it to pick up electrons from the cytochrome b₆/f complex (by way of plastocyanin —PC in the figure) and raise them to a sufficiently high redox potential that, after passing through ferredoxin (Fd in the figure),
• they can reduce NADP⁺ to NADPH.

The sawtooth shifts in redox potential as electrons pass from P₆₈₀ to NADP⁺ have caused this system to be called the Z-Scheme(although as I have drawn the diagram, it looks more like an "N"). It is also called noncyclic photophosphorylation because it produces ATP in a one-way process (unlike cyclic photophosphorylation and pseudocyclic photophosphorylation described below).

More on redox potentials and how they are exploited in photosynthesis.

Link to page analyzing the energy changes that occur during photosynthesis.

Chemiosmosis in Chloroplasts

The energy released as electrons pass down the gradient between photosystem II and plastocyanin (PC) is harnessed by the cytochrome b₆/f complex to pumpprotons (H⁺) against their concentration gradient from the stroma of the chloroplast into the interior of the thylakoid (an example of active transport). As their concentration increases inside (which is the same as saying that the pH of the interior decreases), a strong diffusion gradient is set up. The only exit for these protons is through the ATP synthase complex. As in mitochondria, the energy released as these protons flow down their gradient is harnessed to the synthesis of ATP. The process is called chemiosmosis and is an example of facilitated diffusion.

Link to a description of two experimental tests of chemiosmosis in chloroplasts.

Cyclic Photophosphorylation

• Each CO₂ taken up by the Calvin cycle) requires:
  • 2 NADPH molecules and
  • 3 ATP molecules
• Each molecule of oxygen released by the light reactions supplies the 4 electrons needed to make 2 NADPH molecules.
• The chemiosmosis driven by these 4 electrons as they pass through the cytochrome b₆/f complex liberates only enough energy to pump 12 protons into the interior of the thylakoid.
• But in order to make 3 molecules of ATP, the ATPase in chloroplasts appears to have 14 protons (H⁺) pass through it.
• So there appears to be a deficit of 2 protons.
• How is this deficit to be made up?
• One likely answer: cyclic photophosphorylation.

In cyclic photophosphorylation,

• the electrons expelled by the energy of light absorbed by photosystem I pass, as normal, to ferredoxin (Fd).
• But instead of going on to make NADPH,
• they pass to plastoquinone (PQ) and on back into the cytochrome b₆/f complex.
• Here the energy each electron liberates pumps 2 protons (H⁺) into the interior of the thylakoid — enough to make up the deficit left by noncyclic photophosphorylation.

This process is truly cyclic because no outside source of electrons is required. Like the photocell in a light meter, photosystem I is simply using light to create a flow of current. The only difference is that instead of using the current to move the needle on a light meter, the chloroplast uses the current to help synthesize ATP.

Pseudocyclic Photophosphorylation

Another way to make up the deficit is by a process called pseudocyclic photophosphorylation in which some of the electrons passing to ferredoxin then reduce molecular oxygen back to H₂O instead of reducing NADP⁺ to NADPH.
At first glance, this might seem a fruitless undoing of all the hard work of photosynthesis. But look again. Although the electrons cycle from water to ferredoxin and back again, part of their pathway is through the chemiosmosis-generating stem of cytochrome b₆/f.
Here, then, is another way that simply by turning on a light, enough energy is imparted to electrons that they can bring about the synthesis of ATP.

Antenna Pigments

Chlorophylls a and b differ slightly in the wavelengths of light that they absorb best (although both absorb red and blue much better than yellow and green — View). Carotenoids help fill in the gap by strongly absorbing green light. The entire complex ensures that most of the energy of light will be trapped and passed on to the reaction center chlorophylls.

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Photosynthesis: Pathway of Carbon Fixation

Photosynthesis is the synthesis of organic molecules using the energy of light. For the sugar glucose (one of the most abundant products of photosynthesis) the equation is:
6CO₂ + 12H₂O -> C₆H₁₂O₆ + 6H₂O + 6O₂
A description of the experiments that led to this equation are described in Discovering the Secrets of Photosynthesis.
Light provides the energy to:

• transfer electrons from water to nicotinamide adenine dinucleotide phosphate (NADP⁺) forming NADPH;
• generate ATP.

The details of these processes are described in Photosynthesis: The Role of Light.
ATP and NADPH provide the energy and electrons to reduce carbon dioxide (CO₂) to organic molecules.

The Steps

• CO₂ combines with the phosphorylated 5-carbon sugar ribulose bisphosphate.
• This reaction is catalyzed by the enzyme ribulose bisphosphate carboxylase oxygenase (RUBISCO)(an enzyme which can fairly claim to be the most abundant protein on earth).
• The resulting 6-carbon compound breaks down into two molecules of 3-phosphoglyceric acid (PGA).
• The PGA molecules are further phosphorylated (by ATP) and are reduced (by NADPH) to form phosphoglyceraldehyde (PGAL).
• Phosphoglyceraldehyde serves as the starting material for the synthesis of glucose and fructose.
• Glucose and fructose make the disaccharide sucrose, which travels in solution to other parts of the plant (e.g., fruit, roots).
• Glucose is also the monomer used in the synthesis of the polysaccharides starch and cellulose.

The graphic shows the steps in the fixation of carbon dioxide during photosynthesis. All of these reactions occur in the stroma of the chloroplast.

Link to chloroplasts

These steps were worked out by Melvin Calvin and his colleagues at the University of California and, for this reason, are named the Calvin cycle.

Their Experiment

Apparatus used to follow the fate of 14CO2 in the dark reactions. The algal suspension is placed in the "lollypop", supplied with 14CO2, and illuminated. The dark reactions are halted by draining the contents of the lollypop into a flask of hot alcohol. (Courtesy of Dr. James A. Bassham.)

The experimental apparatus is shown at the right. After various intervals of illumination, a suspension of unicellular algae is inactivated and the contents of the cells extracted. The compounds in a drop of the extract are then separated by paper chromatography.
The identity of each substance may be determined simply by comparing its position with the positions occupied by known substances under the same conditions. Or, a fragment containing the spot can be cut from the sheet and chemically analyzed.
To determine which, if any, of the substances separated on the chromatogram are radioactive, a sheet of X-ray film is placed next to the chromatogram. If dark spots appear on the film (because of radiation emitted by the ¹⁴C atoms), their position can be correlated with the positions of the chemicals in the chromatogram. Using this technique of autoradiography, Calvin found that ¹⁴C turned up in glucose molecules within 30 seconds after the start of photosynthesis. When he permitted photosynthesis to proceed for only 5 seconds, however, the radioactivity was concentrated in several other, smaller, molecules.

The dark spots show the radioactive compounds produced after 10 secs (left) and 2 minutes (right) of photosynthesis by the green alga Scenedesmus. The alga was supplied with carbon dioxide labeled with 14C, a radioactive isotope of carbon. At 10 seconds, most of the radioactivity is found in 3-phosphoglyceric acid ("P-Glyceric"). At 2 minutes, phosphorylated 6-carbon sugars (glucose and fructose) have been synthesized as well as a number of amino acids. The small rectangle and circle (lower right-hand corners) mark the spots where the cell extract was applied. (Courtesy of Dr. James A. Bassham.)

Link to discussion of the free energy changes in photosynthesis.

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Photorespiration and C₄ Plants

All plants carry on photosynthesis by

• adding carbon dioxide (CO₂) to a phosphorylated 5-carbon sugar called ribulose bisphosphate.
• This reaction is catalyzed by the enzyme ribulose bisphosphate carboxylase oxygenase (RUBISCO).
• The resulting 6-carbon compound breaks down into two molecules of 3-phosphoglyceric acid (PGA).
• These 3-carbon molecules serve as the starting material for the synthesis of glucose and other food molecules.
• The process is called the Calvin cycle and the pathway is called the C₃ pathway.

Link to page describing the Calvin cycle.

Photorespiration

As its name suggests, RUBISCO catalyzes two different reactions:

• adding CO₂ to ribulose bisphosphate — the carboxylase activity
• adding O₂ to ribulose bisphosphate — the oxygenase activity.

Which one predominates depends on the relative concentrations of O₂ and CO₂ with

• high CO₂, low O₂ favoring the carboxylase action,
• high O₂, low CO₂ favoring the oxygenase action.

The light reactions of photosynthesis liberate oxygen and more oxygen dissolves in the cytosol of the cell at higher temperatures. Therefore,

• high light intensities and
• high temperatures (above ~ 30°C)

favor the second reaction.

The details of photorespiration

• The uptake of O₂ by RUBISCO forms:
  • the 3-carbon molecule 3-phosphoglyceric acid — just as in the Calvin cycle
  • the 2-carbon molecule glycolate.
• The glycolate enters peroxisomes where it uses O₂ to form intermediates that
• enter mitochondria where they are broken down to CO₂.

So this process uses O₂ and liberates CO₂ as cellular respiration does which is why it is called photorespiration.
It undoes the good anabolic work of photosynthesis, reducing the net productivity of the plant.
For this reason, much effort — so far largely unsuccessful — has gone into attempts to alter crop plants so that they carry on less photorespiration.
The problem may solve itself. If atmospheric CO₂ concentrations continue to rise, perhaps this will enhance the net productivity of the world's crops by reducing losses to photorespiration.

Link to discussion of the earth's carbon cycle.

C₄ Plants

Over 8000 species of angiosperms have developed adaptations which minimize the losses to photorespiration.
They all use a supplementary method of CO₂ uptake which forms a 4-carbon molecule instead of the two 3-carbon molecules of the Calvin cycle. Hence these plants are called C₄ plants. (Plants that have only the Calvin cycle are thus C₃ plants.)

• Some C₄ plants — called CAM plants — separate their C₃ and C₄ cycles by time. CAM plants are discussed below.
• Other C₄ plants have structural changes in their leaf anatomy so that
  • their C₄ and C₃ pathways are separated in different parts of the leaf with
  • RUBISCO sequestered where the CO₂ level is high; the O₂ level low.
  These adaptations are described now.

The details of the C₄ cycle

• After entering through stomata, CO₂ diffuses into a mesophyll cell.
  • Being close to the leaf surface, these cells are exposed to high levels of O₂, but
  • have no RUBISCO so cannot start photorespiration (nor the dark reactions of the Calvin cycle).
• Instead the CO₂ is inserted into a 3-carbon compound (C₃) called phosphoenolpyruvic acid (PEP) forming
• the 4-carbon compound oxaloacetic acid (C₄).
• Oxaloacetic acid is converted into malic acid or aspartic acid (both have 4 carbons), which is
• transported (by plasmodesmata) into a bundle sheath cell. Bundle sheath cells
  • are deep in the leaf so atmospheric oxygen cannot diffuse easily to them;
  • often have thylakoids with reduced photosystem II complexes (the one that produces O₂).
  • Both of these features keep oxygen levels low.
• Here the 4-carbon compound is broken down into
  • carbon dioxide, which enters the Calvin cycle to form sugars and starch.
  • pyruvic acid (C₃), which is transported back to a mesophyll cell where it is converted back into PEP.

These C₄ plants are well adapted to (and likely to be found in) habitats with

• high daytime temperatures
• intense sunlight.

Some examples:

• crabgrass
• corn (maize)
• sugarcane
• sorghum

Although only ~3% of the angiosperms, C₄ plants are responsible for ~25% of all the photosynthesis on land.

C₄ cells in C₃ plants

The ability to use the C₄ pathway has evolved repeatedly in different families of angiosperms — a remarkable example of convergent evolution. Perhaps the potential is in all angiosperms.
A report in the 24 January 2002 issue of Nature (by Julian M. Hibbard and W. Paul Quick) describes the discovery that tobacco, a C₃ plant, has cells capable of fixing carbon dioxide by the C₄ path. These cells are clustered around the veins (containing xylem and phloem) of the stems and also in the petioles of the leaves. In this location, they are far removed from the stomata that could provide atmospheric CO₂. Instead, they get their CO₂ and/or the 4-carbon malic acid in the sap that has been brought up in the xylem from the roots.
If this turns out to be true of many C₃ plants, it would explain why it has been so easy for C₄ plants to evolve from C₃ ancestors.

CAM Plants

These are also C₄ plants but instead of segregating the C₄ and C₃ pathways in different parts of the leaf, they separate them in time instead. (CAM stands for crassulacean acid metabolism because it was first studied in members of the plant family Crassulaceae.)
At night,

• CAM plants take in CO₂ through their open stomata (they tend to have reduced numbers of them).
• The CO₂ joins with PEP to form the 4-carbon oxaloacetic acid.
• This is converted to 4-carbon malic acid that accumulates during the night in the central vacuole of the cells.

In the morning,

• the stomata close (thus conserving moisture as well as reducing the inward diffusion of oxygen).
• The accumulated malic acid leaves the vacuole and is broken down to release CO₂.
• The CO₂ is taken up into the Calvin (C₃) cycle.

These adaptations also enable their owners to thrive in conditions of

• high daytime temperatures
• intense sunlight
• low soil moisture.

Some examples of CAM plants:

• cacti
• Bryophyllum
• the pineapple and all epiphytic bromeliads
• sedums
• the "ice plant" that grows in sandy parts of the scrub forest biome

C₄ Diatoms

On 26 October 2000, Nature reported the discovery of both the C₃ and C₄ pathways in a marine diatom. In this unicellular organism, the two paths are kept separate by having the C₄ path in the cytosol, and the C₃ path confined to the chloroplast. The presence of a C₄ pathway probably reflects the frequent low concentrations of CO₂ in ocean waters.

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