Photosynthesis

Photosynthesis

In this unit of Biology 101 we are going to look at the energy transformations that take place to manufacture the fuel molecules for living organisms, and then at the metabolic processes that cells have to burn fuel to make the ATP needed for all cell work.

The vast majority of living organisms obtain their fuel molecules, directly or indirectly (recycled, so to speak) from the process of photosynthesis. The process of photosynthesis transforms light energy into chemical energy, and uses that energy to produce the carbohydrate, glucose, from water and carbon dioxide molecules.

The majority of autotrophs manufacture their organic molecules by the process of photosynthesis. (Organisms that obtain their organic fuel molecules pre-formed from the environment are heterotrophs. Animals, fungi, many protists and many bacteria are heterotrophs.) Most photosynthetic organisms are plants or protists that contain chlorophyll. Some prokaryotes are also photosynthetic. The Cyanobacteria have chlorophyll pigments; some Bacteria, such as the purple sulfur bacteria, have different light-capturing pigments, and slightly different photosynthetic mechanisms. They are studied in microbiology.

Note: Not all autotrophs are photosynthetic; a tiny proportion of our chemical fuel is manufactured by chemosynthetic organisms. Chemosynthetic autotrophs use energy from chemical reactions involving inorganic atoms and molecules, such as S, Fe, H and N, to make organic compounds. Chemosynthesis is restricted to a very few groups of bacteria, mostly the Archaea. However, chemosynthesis sustains some deep sea-bed ecosystems that surround sulfur vents.

Once organic fuel molecules are manufactured, living organisms must degrade the organic fuel molecules to provide energy to keep their cells alive. These pathways (called cellular respiration) will be discussed later.

The Process of Photosynthesis
As stated, photosynthesis involves the transformation of light energy to chemical energy. The chemical energy is then used to manufacture carbohydrate molecules, primarily glucose. In eukaryotic organisms, and in the Cyanobacteria, the process of photosynthesis also produces oxygen. The photosynthetic bacteria produce organic carbon molecules, but do not produce oxygen. We will discuss the primary photosynthetic processes of plants only.

Photosynthetic Requirements
Photosynthesis occurs in all parts of plants that contain the green pigment, chlorophyll, which is located in the chloroplasts. In most plants, however, photosynthesis occurs mostly in leaves, where chloroplasts are concentrated. In the laboratory, we shall look closely at the leaf structure as it relates to its function on photosynthesis. Let's now turn to this most important process.

Photosynthesis involves two stages, occurring in separate locations within chloroplasts. In the light-dependent reactions of photosynthesis, light energy is transformed into chemical energy in the form of energy transfer molecules in a series of oxidation-reduction reactions that transfer electrons and hydrogen from water to the energy transfer molecule NADP⁺. The light-dependent reactions are known as photophosphorylations, because they involve producing ATP. They take place on the thylakoid membranes of the grana.

In the light-independent reactions or Calvin-Benson cycle (or more simply, the Calvin cycle) the energy from the light reactions is used to manufacture carbohydrate molecules, which form glucose. These reactions occur in the stroma of the chloroplast.

The overall chemical equation for photosynthesis is:

Chlorophyll
6CO₂+ 12H₂0 + 686 kcal ---------> C₆H₁₂OO₆ + 6H₂0+ 6O₂

Chlorophyll
(Carbon dioxide + water + light energy ------> glucose + water + oxygen)

In order to do photosynthesis, water, CO₂, chlorophyll and light energy must be available. We shall briefly look at each of these requirements before we discuss just how this process of photosynthesis works.

Water
Water is the hydrogen and electron donor for the process of photosynthesis.

Water is obtained from the environment, absorbed by roots and conducted throughout the plant in the xylem of the vascular system. Water needed for photosynthesis is but one of the demands for water in plants.

Light energy is used to split water molecules, forming 2H⁺, 2e^-, and Oxygen during the process of photosynthesis.
Carbon Dioxide
Carbon dioxide provides the carbon source for manufacturing the carbohydrates in photosynthesis.

Carbon dioxide diffuses from the atmosphere through pores in leaf surfaces, called stomata, which are formed by a pair of guard cells. The carbon dioxide then diffuses to the photosynthetic cells of the leaf mesophyll. The rate of diffusion of carbon dioxide and availability of carbon dioxide often limit the rate and amount of photosynthesis that occurs in a plant.
Light Waves
Light is a form of electromagnetic radiation. Visible light is a combination of many wavelengths that we can see as different colors (of the rainbow) in the range of 380 - 750 nm. Each wavelength is associated with a specific photon, or particle of energy. In general, shorter wavelengths have more energy.

The light absorbing photosynthetic pigments do not absorb all wavelengths of light equally. Some light energy cannot be absorbed (and is reflected instead) and some is transmitted, or passed through the chloroplasts. The light waves most absorbed and most useful to photosynthesis are reds and blues. Not surprisingly, green light is absorbed poorly.

In the laboratory we shall do an experiment to demonstrate the absorption of different wavelengths of light by the photosynthetic pigments, an absorption spectrum.
The Light Absorbing Pigments of Photosynthesis
Chlorophyll is the primary pigment that absorbs light energy in photosynthesis. In plants, there are two forms of chlorophyll (a, which has a methyl group, and b, which has an aldehyde group) as well as important accessory pigments, the carotenes. Each pigment absorbs certain wavelengths, and collects and concentrates light energy for the photosynthetic process. The red and blue phycocyanin pigments can also absorb light and serve as accessory pigments in photosynthesis.

The Photosystems
The photosynthetic pigment molecules do not work alone. They are arranged on the thylakoids of the chloroplast in clusters of about 300 pigment molecules (plus some other molecules) in a light-harvesting complex (sometimes called the antenna complex) that gathers and transfers energy to a reaction center that has a special chlorophyll a molecule. The reaction centers of Photosystems I and II are activated by slightly different wavelengths of light.

There are two such light-harvesting complexes found in the chloroplasts, called Photosystem I and Photosystem II. In addition to the light-harvesting complexes, each photosystem has a primary electron acceptor, which accepts the electrons released from chlorophyll a, and an electron transport system as well as the.
Chloroplasts
The pigments needed for photosynthesis are located in the chloroplasts. Recall that the chloroplast has a double membrane with a series of internal stacked membranes. Light energy is captured by the pigments located on the special membranes in the chloroplast called thylakoids, which are folded into disk-shaped stacks called grana. The interior compartments of thylakoids serve as reservoirs for hydrogen ions (H⁺) that are needed to produce ATP.

The reactions of photosynthesis that are involved in the transformation of light energy to chemical energy, the light-dependent reactions, occur on the thylakoid membranes of the chloroplast. Each thylakoid has thousands of the two photosystem complexes.

The reactions needed to produce carbohydrates occur in the stroma region of the chloroplast. Enzymes are located here. These reactions are known as the Calvin-Benson cycle or light-independent reactions.
Electron Transport System Molecules (Energy Transfer Molecules)
As discussed previously, many chemical reactions of metabolism are oxidation-reductions that utilize a chain of electron transport molecules.

Electron carriers make it possible for us to trap and use solar energy in photosynthesis. In the process of photosynthesis, the electron transport carriers are embedded in the thylakoid membranes.

The most important energy transfer molecule in photosynthesis is NADP⁺.

The photosynthetic pathways.
The two "stages" of photosynthesis are linked by the products of the first stage, the light-dependent reactions. These products are ATP and NADPH.

Stage I: Light-Dependent Reactions.

The light-dependent reactions transform light energy into chemical energy that is trapped and carried by ATP and NADPH to the Calvin Cycle.
The light-dependent reactions require chlorophyll and occur in the thylakoid membranes of the grana of the chloroplast.
Light energy is also used to split water (Photolysis of water) into:
H₂O -----> 2H⁺ + 2e^- + ¹/₂O₂,
This produces oxygen and provides electrons and Hydrogen for the reduction of NADP to NADPH (NADP gains H⁺ and electrons; the water is oxidized because it loses the H⁺ and e^-)
The light-dependent reactions are photophosphorylations because they involve using light energy to make ATP
The light reactions consume H₂O and produce ATP, NADPH and O₂ as byproduct.

Stage II: Calvin Cycle

These photosynthetic reactions do not use light energy for their energy source. They use the ATP produced in the light reactions for their energy source, and the energy transfer molecule, NADPH to provide Hydrogen and electrons for a high-energy reduction.
Carbohydrate molecules are produced in Calvin Cycle in the stroma of the chloroplast.
In the Calvin cycle, carbon dioxide combines with a 5-carbon sugar (Ribulose bisphosphate) and undergoes a reduction to form 3-carbon molecules. These 3-carbon intermediates can be used to regenerate the 5-carbon sugar or metabolized to form the carbohydrate, glucose.

The Specific Mechanisms of Photosynthesis

The Light Reactions - Cyclic and Non-Cyclic Photophosphorylation)

1. Non-Cyclic Photophosphorylation
Uses

Photosystem I
Photosystem II
Electron Transport System

Inputs

Water
Light energy
Energy Transfer Molecules
ADP and P
NADP⁺

Outputs

ATP
NADPH (reduced form) (from NADP+, the oxidized form)
O₂

Non-Cyclic Light-Dependent Reactions of Photosynthesis

How it works:

Light energy hitting photosystem II excites electrons in the p680 reaction center causing the chlorophyll molecule to lose electrons. They are picked up by the electron carriers and passed “down” slowly releasing energy as they go. Some of this energy is used to produce a H⁺ gradient within the thylakoid. This gradient ultimately drives the mechanism that produces ATP by chemiosmosis (see a bit later).
Light energy is also hitting photosystem I and exciting its reaction center chlorophyll electrons. They are picked up by other electron carriers and transferred to NADP⁺, which will also pick up a H⁺forming NADPH.
The electrons released from photosystem II, once they have passed through the electron transport system, are used to replace the electrons lost by photosystem I that were used to form NADPH.
The electrons lost by photosystem II are replaced by the splitting of water. Water’s electrons are passed to photosystem II; its H⁺ is used in the H⁺ gradient (and can be passed to NADP⁺) and its oxygen is released as oxygen gas molecules.

To summarize, the low energy electrons from water are elevated in energy by passing through both photosystem II and photosystem I and trapped by NADP⁺ where their potential energy will be used for the high energy reduction of carbon in the Calvin cycle. Along the way, ATP, needed for the endergonic Calvin cycle, is also produced.

Orientation of Electron Transport Molecules and Photosystems in the Thylakoid Membranes

2. Cyclic Photophosphorylation
Uses

Photosystem I
Electron Transport System

Inputs

Light energy

Outputs

ATP (from ADP and P)

Cyclic Photophosphorylation only uses Photosystem I and produces ATP. In this process, electrons released from the chlorophyll p700 are returned back to Photosystem I after passing through the chain of electron carriers.

Cyclic Photophosphorylation

Before we go much further, just how is the ATP, produced in the light-dependent reactions really made?

The Chemiosmotic Theory of ATP Synthesis
We have discussed that electrons released from molecules (such as chlorophyll) can travel down an electron transport system, releasing their energy in controlled bits. This energy, as we have said, can be used for the synthesis of ATP.

In photosynthesis, the molecules of electron transport system are located in the thylakoid membrane. The energy released from the electron transport system is used to move Hydrogen ions (H⁺) from the stroma into the inner thylakoid compartments by active transport. This concentration of H⁺ in the inner compartment of the thylakoid establishes a concentration and a charge gradient in the thylakoid compartment that has a high potential energy.

The accumulated H⁺ ions diffuse through ATP synthase protein complex channels in the thylakoid membranes that are coupled to ATP synthesis. As the H⁺ ions flow down the gradient in the protein channels, their energy is used to make ATP from ADP and P on the other side of the thylakoid membrane in the stroma.

Note: Peter Mitchell won a Nobel prize in 1978 for determining the chemiosmotic theory of ATP synthesis. ATP is synthesized in the mitochondria during cell respiration by a similar mechanism.

Chemiosmotic synthesis of ATP in the Thylakoids

Chemiosmosis in Photosynthesis - a second view

The Calvin-Benson Cycle and Carbon Fixation
The second set of reactions for photosynthesis is known as the Calvin cycle, or the light-independent reactions. They occur in the stroma region of the chloroplast and use the products formed during the light reactions of photosynthesis.

There are many parts to the Calvin cycle:

Carbon Fixation
Reduction
Regeneration
Surplus (Output)

The requirements for the Calvin cycle are:

Carbon dioxide (CO₂)
NADPH from the light-dependent reactions (reducing power, source of electrons and the hydrogen source)
ATP from the light-dependent reactions (energy source)
Ribulose bisphosphate, regenerated in the cycle
Appropriate enzymes for each step in the cycle. Of these, Ribulose bisphosphate carboxylase (Rubisco) is especially important.

The Metabolic Intermediate in Process:

G3P (Glyceraldehyde 3 Phosphate)

The Calvin cycle produces:

Glucose (carbohydrate)
Ribulose bisphosphate, regenerated in the cycle

By the way, the process we are discussing was determined using radioisotopes of ¹⁴C. The researchers (Calvin, Benson and others) won a Nobel prize for this. This pathway is called 3-carbon photosynthesis because of the 3-C G3P intermediate to distinguish it from an alternative pathway known as 4-carbon photosynthesis, to be discussed in a bit. To some extent, the Calvin cycle looks like a carbon cut-and-paste dance. Well, it is. It's easy to follow the maze if one counts carbons, even in more detail than presented in your text. It also helps to remember that without this happening, you'd starve!

Note: Only the carbon backbone of the molecules are shown in the diagrams, except where other atoms are critical to the structure!

Carbon Fixation and the Reduction Phase of the Calvin Cycle
This must repeat 6 times to form 1 glucose

CO2   +   RuBP (Ribulose bisphosphate) ----> Hexose Phosphate ---->

C     +   C                           ---->     C            ---->
          C                                     C
          C                                     C
          C                                     C
          C-P                                   C
                                                C-P

2 PGA (Phosphoglyceric Acid) ----> 2 G3P* (Glyceraldehyde 3 Phosphate)
 (+ 2  ATP  +  2 NADPH)      ---->   (+ 2 P  +  2 ADP  +  2 NADP+)

2 of C                       ---->         2 of     C
     C                                              C
     C-P                                            C-P

A balanced Reduction uses: 6CO2 + 6 RuBP + 12 ATP + 12 NADPH to produce 12 G3P.

* Although not diagrammed, the G3P has 1 more H atom in its structure than PGA. The H was donated by NADPH during the reduction step. This forms an aldehyde molecule, rather than an organic acid. Molecular diagrams are available on request.

Regeneration of Ribulose bisphosphate (RuBp) phase
This will use 10 of the 12 G3P molecules from the reduction phase.

10 G3P + 6 ATP ----> 6 RuBP + 6 H2O (+ 6 ADP)

C                                                     C
C                                                     C
C                                                     C     + ATP ---->  RuBP
                                                      C
                                                      C
C
C                C
C                C     remove "head"
                 C
                 C
C                C          C                         C
C                C          C                         C
C                           C                         C     + ATP ---->  RuBP
                            C                         C
                            C     remove "head"       C
C                           C
C                           C
C
                                                      C
                                                      C
C                                                     C     + ATP ---->  RuBP
C                                                     C
C                                                     C

C                                                     C
C                                                     C
C                                                     C     + ATP ---->  RuBP
                                                      C
                                                      C
C
C                C
C                C     remove "head"
                 C
                 C
C                C          C                         C
C                C          C                         C
C                           C                         C     + ATP ---->  RuBP
                            C                         C
                            C     remove "head"       C
C                           C
C                           C
C
                                                      C
                                                      C
C                                                     C     + ATP ---->  RuBP
C                                                     C
C                                                     C

Note: Water, ADP and P are given off in the process.

The Surplus Phase - Producing Glucose
The remaining 2 molecules of G3P are converted into one Glucose molecule

2 G3P ------------------------------------------------> 1 Glucose

1.                2.           3.           4.            5.

C   +   C   ----> C-P    ----> C     -----> C=O     ----> C=O
C       C         C            C=O          C             C
C-P     C-P       C            C            C             C
                  C            C            C             C
                  C            C            C             C
                  C-P          C-P          C-P           C

                              (+ P)                      (+ P)

1. G3P (Glyceraldehyde 3 Phosphate)
2. Fructose 1,6 bisphosphate
3. Fructose 6 phosphate
4. Glucose 6 phosphate
5. Glucose

Note The =O is shown only to indicate the isomer rearrangement that occurs in step 3 to 4 between fructose-6-phosphate and glucose-6-phosphate.

What comes after Photosynthesis?
Although glucose is the typical end product of photosynthesis, and as we shall discuss, the primary fuel molecule for living organisms, plants are capable of synthesizing all of their organic molecules from photosynthetic intermediates, notably G3P and glucose phosphate. Plants are more versatile in their synthetic abilities than are animals. In addition there are whole groups of plant products, called secondary metabolites, synthesized directly or perhaps as by-products of plant activity.

Some of these metabolites are protective in nature, such as toxins; some, such as lignin, are used in plant structure.

How Productive is Photosynthesis?
We have seen that it takes 18 ATP and 12 NADPH to make one molecule of glucose. Much of the light energy that hits the surface of plants is not absorbed and not available. And much of the light that hits earth does not hit photosynthetic surfaces of plants.

Plants also need water and carbon dioxide for photosynthesis. The stomata found in the leaf surface that permit CO₂ to diffuse into the leaf also permit the diffusion of water and O₂ out of the leaf. This loss of water can be significant. As much as 90% of the water absorbed by the plant is lost this way. (This evaporation of water through the stomata is also used by the plant to generate a tension that serves to pull water up through the xylem from the roots to stems and leaves, so it is not all a negative thing for the plant.)

Photorespiration
Under very hot and dry conditions, many plants must close their stomata to minimize water loss. During these times the ratio of oxygen to carbon dioxide in the leaf increases, and this favors a process called photorespiration.

Rubisco, the enzyme that brings CO₂ and RuBP together, works only when the concentration of CO₂ is high relative to the level of O₂. When CO₂ levels drop, the enzyme, Rubisco, combines RuBP with O₂ and the Calvin cycle is disrupted. Photorespiration decreases the photosynthetic output of the plant.

C-4 Photosynthesis
Some plants in hot, dry environments have evolved mechanisms to minimize photorespiration. When CO₂ diffuses into a mesophyll cell, it is combined with a 3-carbon compound, PEP (phosphoenolpyruvate), forming a 4-carbon acid, Oxaloacetic acid.

This 4-carbon acid is then transferred to the bundle sheath cells of the leaf. This is a more efficient trap for carbon dioxide since the 4-carbon acid can accumulate during non-light periods, concentrating carbon dioxide when photosynthesis cannot occur, and can be used during periods of low moisture when stomata are closed to prevent water loss. Such plants have a photosynthetic mechanism called C-4 photosynthesis (because of the 4-carbon acids formed).

C-4 plants also perform the two stages of photosynthesis in separate cells to keep O2 away from Rubisco. The light reactions (which produce oxygen) occur in the leaf mesophyll cells that surround the bundle sheath cells of veins. The Calvin cycle occurs in specialized bundle sheath cells whose chloroplasts have very few thylakoids.

C-4 photosynthesis is highly productive in hot and dry environments. The world's most productive plants are C-4 plants. However, regenerating PEP requires ATP, so C-4 photosynthesis is not always more productive than the C-3 pathway. Unfortunately, the pathway is genetic, so plants can't choose.

CAM - Another Conservation Mechanism
Some other plants minimize water loss by reversing the time of day when stomates are open. Plants that have reverse stomatal opening can also store CO₂ in 4-carbon acids. They do not, however, have photosynthesis separated into two different cells. In the daytime, this CO₂ is released for "normal" C-3 photosynthesis, while the stomata can remain closed to prevent excessive water loss. The name, CAM (for Crassulacean Acid Metabolism) is derived from the types of plants in which it was first discovered, Crassuleans, and for the fact the CO₂ trapped forms acids.

Bacterial Photosynthesis
Some bacteria have photosynthetic pigments and a process of photosynthesis. There are some differences, however:

Bacterial chlorophyll is different from the chlorophyll of other photosynthetic organisms.
Bacteria do a just cyclic photophosphorylation. They can not do photolysis, and do not produce oxygen.

Chemosynthetic Bacteria
As mentioned in the introduction to this chapter, some bacteria can use inorganic molecules such as Fe⁺⁺, NH₄⁺, S and H to provide energy to "fix" carbon (that is make organic compounds from inorganic sources). In the scheme of life, chemosynthesis plays a small part in energy acquisition. Yet the environmental role of chemosynthetic bacteria, such as in the nitrogen cycle, is critical.

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