13.25: S2023_Bis2A_Singer_Photosynthesis

Light Energy and Pigments

Light Energy

The sun emits an enormous amount of electromagnetic radiation (solar energy) that spans a broad swath of the electromagnetic spectrum, the range of all radiation frequencies. When solar radiation reaches Earth, a fraction of this energy interacts with and may transfer to the matter on the planet. This energy transfer results in a wide variety of different phenomena, from influencing weather patterns to driving myriad biological processes. In BIS2A, we are largely concerned with the latter, and below, we discuss some very basic concepts related to light and its interaction with biology.

First, however we need to refresh a few key properties of light:

1. Light in a vacuum travels at a constant speed of 299,792,458 m/s. We often abbreviate the speed of light with the variable "c".
2. Light has properties of waves. A specific "color" of light has a characteristic wavelength.

We refer to the distance between peaks in a wave as the wavelength and abbreviate it with the Greek letter lambda (Ⲗ). Attribution: Marc T. Facciotti (original work)

The inverse proportionality of frequency and wavelength. Wave 1 has a wavelength that is 2x that of wave 2 (Ⲗ1 > Ⲗ2). If the two waves are traveling at the same speed (c)—imagine that both of the whole lines that are dragged past the fixed vertical line at the same speed—then the number of times a wave peak passes a fixed point is greater for wave 2 than wave 1 (f2 > f1). Attribution: Marc T. Facciotti (original work)

 

3. Finally, each frequency (or wavelength) of light is associated with a specific energy. We'll call energy "E". The relationship between frequency and energy is:

\[E = h \times f\]

where h is a constant called the Planck constant (~6.626x10^-34 Joule•second when frequency is expressed in cycles per second). Given the relationship between frequency and wavelength, you can also write E = h*c/Ⲗ. Therefore, the larger the frequency (or shorter the wavelength), the more energy is associated with a specific "color". Wave 2 in the figure above is associated with greater energy than wave 1.

 

The sun emits energy in the form of electromagnetic radiation. All electromagnetic radiation, including visible light, is characterized by its wavelength. The longer the wavelength, the less energy it carries. The shorter the wavelength, the more energy is associated with that band of the electromagnetic spectrum.

The Light We See

The visible light seen by humans as white light is composed of a rainbow of colors, each with a characteristic wavelength. Certain objects, such as a prism or a drop of water, disperse white light to reveal the colors to the human eye. In the visible spectrum, violet and blue light have shorter (higher energy) wavelengths while the orange and red light have a longer (lower energy) wavelengths.

The colors of visible light do not carry the same amount of energy. Violet has the shortest wavelength and therefore carries the most energy, whereas red has the longest wavelength and carries the least amount of energy. Credit: modification of work by NASA

 

Absorption by Pigments

The interaction between light and biological systems occurs through several mechanisms, some of which you may learn about in upper division courses in cellular physiology or biophysical chemistry. In BIS2A, we concern ourselves mostly with the interaction between light and biological pigments. These interactions can start a variety of light-dependent biological processes that can be grossly grouped into two functional categories: cellular signaling and energy harvesting. Signaling molecules perceive changes in the environment (in this case, changes in light). An example of a signaling interaction might be the interaction between light and the pigments expressed in an eye. Light/pigment interactions that are involved in energy harvesting are used for—not surprisingly—capturing the energy in the light and transferring it to the cell to fuel biological processes. Photophosphorylation, which we will learn more about soon, is one example of an energy harvesting interaction.

 

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Possible NB Discussion Point

Photophosphorylation is a process involving an electron transport chain that allows organisms to harvest energy from light. Some of you may already be familiar with this process. Many of you are learning about this for the first time. Given your current knowledge base, offer your best explanation or hypothesis as to how light interacts with the ETC. You will have a chance to revisit this topic very soon.

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At the center of the biological interactions with light are groups of molecules we call organic pigments. Whether in the human retina, chloroplast thylakoid, or microbial membrane, organic pigments often have specific ranges of energy or wavelengths that they can absorb. The sensitivity of these molecules for different wavelengths of light is due to their unique chemical makeups and structures. A range of the electromagnetic spectrum is given a couple of special names because of the sensitivity of some key biological pigments: The retinal pigment in our eyes, when coupled with an opsin sensor protein, “sees” (absorbs) light predominantly between the wavelengths between of 700 nm and 400 nm. Because this range defines the physical limits of the electromagnetic spectrum that we can actually see with our eyes, we refer to this wavelength range as the “visible range”. For similar reasons, as plants pigment molecules tend to absorb wavelengths of light mostly between 700 nm and 400 nm, plant physiologists refer to this range of wavelengths as "photosynthetically active radiation".

 

Three Key Types of Pigments Commonly Discussed in General Biology

Chlorophylls

Chlorophylls (including bacteriochlorophylls) are part of a large family of pigment molecules. There are five major chlorophyll pigments named: a, b, c, d, and f. Chlorophyll a is related to a class of more ancient molecules found in bacteria called bacteriochlorophylls. Chlorophylls are structurally characterized by ring-like porphyrin group that coordinates a metal ion. This ring structure is chemically related to the structure of heme compounds that also coordinate a metal and are involved in oxygen binding and/or transport in many organisms. We distinguish different chlorophylls from one another by different "decorations"/chemical groups on the porphyrin ring.

The structure of heme and chlorophyll a molecules. The common porphyrin ring is colored in red. Attribution: Marc T. Facciotti (original work)

Carotenoids

Carotenoids are the red/orange/yellow pigments found in nature. They are found in fruit—the red of tomato (lycopene), the yellow of corn seeds (zeaxanthin), or the orange of an orange peel (β-carotene)—which serve as biological "advertisements" to attract seed dispersers (animals or insects that may carry seeds elsewhere). In photosynthesis, carotenoids function as photosynthetic pigments. In addition, when a leaf is exposed to full sun, that surface is required to process an enormous amount of energy; If that energy is not managed properly, it can do significant damage. Therefore, many carotenoids help absorb excess energy in light and safely dissipate that energy as heat.

Flavonoids

Flavonoids are a very broad class of compounds that are found in great diversity in plants. These molecules come in many forms but all share a common core structure shown below. The diversity of flavonoids comes from the many combinations of functional groups that can "decorate" the core flavone.

The core ring structure of flavans.

Each type of pigment can be identified by the specific pattern of wavelengths it absorbs from visible light. We define this characteristic as the pigment's absorption spectrum. The graph in the figure below shows the absorption spectra for chlorophyll a, chlorophyll b, and a type of carotenoid pigment called β-carotene (which absorbs blue and green light). Notice how each pigment has a distinct set of peaks and troughs, revealing a highly specific pattern of absorption. These differences in absorbance are because of differences in chemical structure (some of these are highlighted in the figure). Chlorophyll a absorbs wavelengths from either end of the visible spectrum (blue and red), but not green. Because chlorophyll reflects green light and absorbs other wavelengths of light, things containing this pigment appear green. Carotenoids absorb in the short-wavelength blue region and reflect the longer yellow, red, and orange wavelengths.

(a) Chlorophyll a, (b) chlorophyll b, and (c) β-carotene are hydrophobic organic pigments found in the thylakoid membrane. Chlorophyll a and b, which are identical except for the part indicated in the red box, are responsible for the green color of leaves. Note how the small amount of difference in chemical composition between different chlorophylls leads to different absorption spectra. β-carotene is responsible for the orange color in carrots. Each pigment has a unique absorbance spectrum (d).

Importance of having multiple different pigments

Not all photosynthetic organisms have full access to sunlight. Some organisms grow underwater where light intensity and the number of wavelengths decrease and change, respectively, with depth. Other organisms grow in competition for light. For instance, plants on the rainforest floor must be able to absorb any bit of light that comes through because the taller trees absorb most of the sunlight and scatter the remaining solar radiation. To account for these variable light conditions, many photosynthetic organisms have a mixture of pigments whose expression they can tune to improve the organism's ability to absorb energy from a wider range of wavelengths than would be possible with one pigment alone.

 

Photophosphorylation

Photophosphorylation an overview

Photophosphorylation is the process of transferring the energy from light into chemicals, particularly ATP. The evolutionary roots of photophosphorylation are likely in the anaerobic world, between 3 billion and 1.5 billion years ago, when life was abundant in the absence of molecular oxygen. Photophosphorylation probably evolved relatively shortly after electron transport chains (ETC) and anaerobic respiration provided metabolic diversity. The first step of the process involves the absorption of a photon by a pigment molecule. Light energy transfers to the pigment and promotes electrons (e^-) into a higher quantum energy state—something biologists term an "excited state". Note the use of anthropomorphism here; the electrons are not "excited" in the classic sense and aren't suddenly hopping all over or celebrating their promotion. They are simply in a higher energy quantum state. In this state, the electrons are colloquially said to be "energized". While in the "excited" state, the pigment now has a much lower reduction potential and can donate the "excited" electrons to other carriers with greater reduction potentials. These electron acceptors may become donors to other molecules with greater reduction potentials and, in doing so, form an electron transport chain.

As electrons pass from one electron carrier to another via red/ox reactions, enzymes can couple these exergonic electron transfers to the endergonic transport (or pumping) of protons across a membrane to create an electrochemical gradient. This electrochemical gradient generates a proton motive force (PMF).  Enzymes can couple the exergonic drive of these protons to reach equilibrium to the endergonic production of ATP, via ATP synthase. As we will see in more detail, the electrons involved in this electron transport chain can have one of two fates: (1) they may return to their initial source in a process called cyclic photophosphorylation; or (2) they can transfer onto a close relative of NAD⁺ called NADP⁺. If electrons return to the original pigment in a cyclic process, the whole process can start over. If, however, the electron transfers onto NADP⁺ to form NADPH (**shortcut note—we didn't explicitly mention any protons but assume that they are also involved**), the original pigment must regain an electron from somewhere else. This electron must come from a source with a smaller reduction potential than the oxidized pigment and depending on the system there are different sources, including H₂O, reduced sulfur compounds such as SH₂ and even elemental S⁰.

What happens when a compound absorbs a photon of light?

When a compound absorbs a photon of light, the compound is said to leave its ground state and become "excited".

Figure 1. A diagram depicting what happens to a molecule that absorbs a photon of light. Attribution: Marc T. Facciotti (original work)

What are the fates of the "excited" electron? There are four possible outcomes, which are schematically diagrammed in the figure below. These options are:

1. The e^- can relax to a lower quantum state, transferring energy as heat.
2. The e^- can relax to a lower quantum state and transfer energy into a photon of light—a process known as fluorescence.
3. The energy can be transferred by resonance to a neighboring molecule as the e^- returns to a lower quantum state.
4. The energy can change the reduction potential such that the molecule can become an e^- donor. Linking this excited e^- donor to a proper e^- acceptor can lead to an exergonic electron transfer. The excited state can be involved in red/ox reactions.

Figure 2. What can happen to the energy absorbed by a molecule.

As the excited electron decays back to its lower energy state, it can transfer its energy in a variety of ways. While many so-called antenna or auxiliary pigments absorb light energy and transfer it to something known as a reaction center (by mechanisms depicted in option III in Figure 2), it is what happens at the reaction center that we are most concerned with (option IV in the figure above). Here a chlorophyll or bacteriochlorophyll molecule absorbs a photon's energy, and an electron is excited. This energy transfer suffices to allow the reaction center to donate the electron in a red/ox reaction to a second molecule. This starts the electron transport reactions. The result is an oxidized reaction center that must now be reduced to start the process again. How this happens is the basis of electron flow in photophosphorylation and we describe this below.

 

Simple photophosphorylation systems: anoxygenic photophosphorylation

Early in the evolution of photophosphorylation, these reactions evolved in anaerobic environments where there was very little molecular oxygen available. Two sets of reactions evolved under these conditions, both directly from anaerobic respiratory chains as described previously. We know these as the light reactions because they require the activation of an electron (an "excited" electron) from the absorption of a photon of light by a reaction center pigment, such as bacteriochlorophyll. We classify the light reactions either as cyclic or as noncyclic photophosphorylation, depending upon the final state of the electron(s) removed from the reaction center pigments. If the electron(s) returns to the original pigment reaction center, such as bacteriochlorophyll, this is cyclic photophosphorylation; the electrons make a complete circuit. We diagram this in Figure 4. If the electron(s) are used to reduce NADP⁺ to NADPH, the electron(s) are removed from the pathway and end up on NADPH; we refer to this process as noncyclic since the electrons are no longer part of the circuit. Here the reaction center must be re-reduced before the process can happen again. Therefore, an external electron source is required for noncylic photophosphorylation. In these systems reduced forms of Sulfur, such as H₂S, which can be used as an electron donor and is diagrammed in Figure 5. To help you better understand the similarities of photophosphorylation to respiration, we provided a red/ox tower that contains many commonly used compounds involved with photosphosphorylation.

 

oxidized form	reduced form	n (electrons)	Eo´ (volts)
PS1* (ox)	PS1* (red)	-	-1.20
ferredoxin (ox) version 1	ferredoxin (red) version 1	1	-0.7
PSII* (ox)	PSII* (red)	-	-0.67
P840*(ox)	PS840*(red)	-	-0.67
acetate	acetaldehyde	2	-0.6
CO2	Glucose	24	-0.43
ferredoxin (ox) version 2	ferredoxin (red) version 2	1	-0.43
CO2	formate	2	-0.42
2H^+	H2	2	-0.42 (at [H^+] = 10^-7; pH=7)
NAD^++ 2H^+	NADH + H^+	2	-0.32
NADP^++ 2H^+	NADPH + H^+	2	-0.32
Complex I FMN (enzyme bound)	FMNH2	2	-0.3
Lipoic acid, (ox)	Lipoic acid, (red)	2	-0.29
FAD^+ (free) + 2H^+	FADH2	2	-0.22
Pyruvate + 2H^+	lactate	2	-0.19
FAD^+ + 2H^+ (bound)	FADH2 (bound)	2	0.003-0.09
CoQ (Ubiquinone - UQ + H^+)	UQH.	1	0.031
UQ + 2H^+	UQH2	2	0.06
Plastoquinone; (ox)	Plastoquinone; (red)	-	0.08
Ubiquinone; (ox)	Ubiquinone; (red)	2	0.1
Complex III Cytochrome b2; Fe^3+	Cytochrome b2; Fe^2+	1	0.12
Complex III Cytochrome c1; Fe^3+	Cytochrome c1; Fe^2+	1	0.22
Cytochrome c; Fe^3+	Cytochrome c; Fe^2+	1	0.25
Complex IV Cytochrome a; Fe^3+	Cytochrome a; Fe^2+	1	0.29
1/2 O2 + H2O	H2O2	2	0.3
P840^GS(ox)	PS840^GS(red)	-	0.33
Complex IV Cytochrome a3; Fe^3+	Cytochrome a3; Fe^2+	1	0.35
Ferricyanide	ferrocyanide	2	0.36
Cytochrome f; Fe^3+	Cytochrome f; Fe^2+	1	0.37
PSI^GS(ox)	PSI^GS(red)	.	0.37
Nitrate	nitrite	1	0.42
Fe^3+	Fe^2+	1	0.77
1/2 O2 + 2H^+	H2O	2	0.816
PSII^GS(ox)	PSII^GS(red)	-	1.10
* Excited State, after absorbing a photon of light GS Ground State, state prior to absorbing a photon of light PS1: Oxygenic photosystem I P840: Bacterial reaction center containing bacteriochlorophyll (anoxygenic) PSII: Oxygenic photosystem II

Figure 3. Electron tower that has a variety of common photophosphorylation components. PSI and PSII refer to Photosystems I and II of the oxygenic photophosphorylation pathways.

 

Cyclic photophosphorylation

In cyclic photophosphorylation the bacteriochlorophyll_red molecule absorbs enough light energy to energize and eject an electron to form bacteriochlorophyll_ox. The electron reduces a carrier molecule in the reaction center which in turn reduces a series of carriers via red/ox reactions. These carriers are the same carriers found in respiration. If the change in reduction potential from the various red/ox reactions are sufficiently large, H⁺ protons can be translocated across a membrane. Eventually, the electron is used to reduce bacteriochlorophyll_ox (making a complete loop) and the whole process can start again. This flow of electrons is cyclic and is therefore said to drive a processed called cyclic photophosphorylation. The electrons make a complete cycle: bacteriochlorophyll is the initial source of electrons and is the final electron acceptor. ATP is produced via the F₁F₀ ATPase. The schematic in Figure 4 demonstrates how cyclic electrons flow and thus how cyclic photophosphorylation works.

Figure 4. Cyclic electron flow. The reaction center P840 absorbs light energy and becomes excited, denoted with an *. The excited electron is ejected and used to reduce an FeS protein leaving an oxidized reaction center. The electron its transferred to a quinone, then to a series of cytochromes, which in turn reduces the P840 reaction center. The process is cyclical. Note the gray array coming from the FeS protein going to a ferridoxin (Fd), also in gray. This represents an alternative pathway the electron can take and will be discussed below in noncyclic photophosphorylation. Note that the electron that initially leaves the P840 reaction center is not necessarily the same electron that eventually finds its way back to reduce the oxidized P840.

 

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Possible NB Discussion Point

The figure of cyclic photophosphorylation above depicts the flow of electrons in a respiratory chain. How does this process help generate ATP? Why might running the process in a cyclical fashion be advantageous for a cell?

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Noncyclic photophosphorylation

In cyclic photophosphorylation, electrons cycle from bacteriochlorophyll (or chlorophyll) to a series of electron carriers and eventually back to bacteriochlorophyll (or chlorophyll); there is theoretically no net loss of electrons and they stay in the system. In noncyclic photophosphorylation, electrons leave from the photosystem and red/ox chain and eventually end up on NADPH. That means there needs to be a source of electrons, a source that has a smaller reduction potential than bacteriochlorophyll (or chlorophyll) that can donate electrons to bacteriochlorophyll_ox to reduce it. From looking at the electron tower in Figure 3, you can see what compounds can reduce the oxidized form of bacteriochlorophyll. The second requirement is that, when bacteriochlorophyll becomes oxidized by ejecting its excited electron, it must reduce a carrier that has a greater reduction potential than NADP/NADPH (see the electron tower). Here, electrons can flow from energized bacteriochlorophyll to NADP forming NADPH and oxidized bacteriochlorophyll. The system loses electrons and they end up on NADPH; to complete the circuit, bacteriochlorophyll_ox is reduced by an external electron donor such as H₂S or elemental S⁰.

Noncyclic electron flow

Figure 5. Noncyclic electron flow. In this example, the P840 reaction center absorbs light energy and becomes energized; the emitted electron reduces a FeS protein and reduces ferridoxin. Reduced ferridoxin (Fd_red) can now reduce NADP to form NADPH. The electrons are now removed from the system, finding their way to NADPH. The electrons need to be replaced on P840, which requires an external electron donor. Here, H₂S serves as the electron donor.

 

We note that for bacterial photophosphorylation pathways, for each electron donated from a reaction center [remember only one electron is actually donated to the reaction center (or chlorophyl molecule)], the resulting output from that electron transport chain is either  the formation of NADPH (requires two electrons) or ATP can be made but NOT not both. The path the electrons take in the ETC can have one or two outcomes. This puts limits on the versatility of the bacterial anoxygenic photosynthetic systems. But what would happen if a process evolved that used both systems?  More precicely, a cyclic and noncyclic photosynthetic pathway which could form both ATP and NADPH from a single input of electrons? A second limitation is that these bacterial systems require compounds such as reduced sulfur to act as electron donors to reduce the oxidized reaction centers, but they are not necessarily widely found compounds. What would happen if a chlorophyll _ox molecule would have a reduction potential higher (more positive) than that of the molecular O₂/H₂O reaction? Answer: a planetary game changer.

 

Oxygenic photophosphorylation

Generation of NADPH and ATP

The overall function of light-dependent reactions is to transfer solar energy into chemical compounds, largely the molecules NADPH and ATP. This energy supports the light-independent reactions and fuels the assembly of sugar molecules. We depict the light-dependent reactions in Figures 6 and 7. Protein complexes and pigment molecules work together to produce NADPH and ATP.

Figure 1. A photosystem consists of a light-harvesting complex and a reaction center. Pigments in the light-harvesting complex pass light energy to two special chlorophyll a molecules in the reaction center. The light excites an electron from the chlorophyll a pair, which passes to the primary electron acceptor. The excited electron must then be replaced. In (a) photosystem II, the electron comes from the splitting of water, which releases oxygen as a waste product. In (b) photosystem I, the electron comes from the chloroplast electron transport chain discussed below.

The actual step that transfers light energy into a biomolecule takes place in a multiprotein complex called a photosystem, two types of which are found embedded in the thylakoid membrane, photosystem II (PSII) and photosystem I (PSI). The two complexes differ based on what they oxidize (that is, the source of the low energy electron supply) and what they reduce (the place to which they deliver their energized electrons).

Both photosystems have the same basic structure; several antenna proteins to which the chlorophyll molecules bind surround the reaction center in which the photochemistry takes place. Each photosystem associates with the light-harvesting complex, which passes energy captured from sunlight to the reaction center; it comprises multiple antenna proteins that contain a mixture of 300–400 chlorophyll a and b molecules and other pigments like carotenoids. The absorption of a single photon—a distinct quantity or “packet” of light—by any of the chlorophylls pushes that molecule into an excited state. In short, the biological molecule has now captured light energy. However, the energy is not yet stored in any useful form. The captured energy transfers from chlorophyll to chlorophyll until, eventually (after about a millionth of a second), it arrives to the reaction center. Up to this point, only energy has been transferred between molecules, not electrons.

Figure 2. In the photosystem II (PSII) reaction center, energy from sunlight is used to extract electrons from water. The electrons travel through the chloroplast electron transport chain to photosystem I (PSI), which reduces NADP⁺ to NADPH. The electron transport chain moves protons across the thylakoid membrane into the lumen. At the same time, splitting of water adds protons to the lumen, and reduction of NADPH removes protons from the stroma. The net result is a low pH in the thylakoid lumen, and a high pH in the stroma. ATP synthase uses this electrochemical gradient to make ATP.

The reaction center contains a pair of chlorophyll a molecules with a special property. Those two chlorophylls can undergo oxidation upon excitation; they can give up an electron in a process called a photoactivation. It is at this step in the reaction center, this step in photophosphorylation, that light energy transfers into an excited electron. All the subsequent steps involve a getting of that electron onto the energy carrier NADPH for delivery to the Calvin cycle where the electron can be deposited onto carbon for long-term storage in the form of a carbohydrate.

 

The Z-scheme

PSII and PSI are two major components of the photosynthetic electron transport chain, which also include the cytochrome complex. The reaction center of PSII (called P680) delivers its high-energy electrons, one at a time, to a primary electron acceptor called pheophytin (Ph), and then sequentially to two bound plastoquinones Q_A and Q_B. Electrons then transfer off of PSII onto a pool of mobile plastoquinones (Q pool) which then transfer the electrons to a protein complex called Cytochromeb₆f. The cytochrome complex uses the red/ox transfers to pump proton across the thylakoyd membrane establishing a proton-motive force that can be used for the synthesis of ATP. Electrons leaving the Cytochrome transfer to a copper-containing protein called plastocyanin (PC) which then transfers electrons to PSI (P700). P680’s missing electron is replaced by extracting an electron from water; thus, water splits and PSII is re-reduced after every photoactivation step. Just for the sake of sharing some numbers: Splitting one H₂O molecule releases two electrons, two hydrogen atoms, and one atom of oxygen. The formation of one molecule of diatomic O₂ gas requires the splitting two water molecules. In plant tissue, mitochondria use about ten percent of that oxygen to support oxidative phosphorylation. The rest escapes to the atmosphere where it is used by aerobic organisms to support respiration.

As electrons move through the proteins that live between PSII and PSI, they take part in exergonic red/ox transfers. The free energy associated with the exergonic red/ox reaction is coupled to the endergonic transport of protons from the stromal side of the membrane to the thylakoid lumen by the cytochrome complex. Those hydrogen ions, plus the ones produced by splitting water, accumulate in the thylakoid lumen and create a proton motive force that will drive the synthesis of ATP in a later step. Since the electrons on PSI now have a greater reduction potential than when they started their trek (it is important to note that unexcited PSI has a greater red/ox potential than NADP⁺/NADPH), they must be re-energized in PSI before getting deposited onto NADP⁺. Therefore, to complete this process, another photon must be absorbed by the PSI antenna. That energy transfers to the PSI reaction center (called P700). P700 then oxidizes and sends an electron through several intermediate red/ox steps to NADP⁺ to form NADPH. Thus, PSII captures the energy in light and couples its transfer via red/ox reactions to the creation of a proton gradient. As already noted, the exergonic and controlled relaxation of this gradient can be coupled to the synthesis of ATP. PSI captures energy in light and couples that, through a series of red/ox reactions, to reduce NADP⁺ into NADPH. The two photosystems work in concert, in part, to guarantee that the production of NADPH will be in the right proportion to the production of ATP. Other mechanisms exist to fine tune that ratio to match the chloroplast’s constantly changing energy needs.

Figure 3. A diagram depicting the flow of electrons and the red/ox potentials of their carriers in oxygenic photosynthetic systems expressing both photosystem I (boxed in blue) and photosystem II (boxed in green). Ph = pheophytin; Q_{A =} bound plastoquinone, Q_B = more loosely associated plastoquinone; Q pool = mobile plastoquinone pool; Cyt bf = Cytochrome b₆f complex; PC = plastocyanin; Chl_a0 = special chrolophyl; A₁ = vitamin K; F_x and F_AB = iron-sulfur centers; F_d = ferredoxin; FNR = ferredoxin-NADP reductase. Attribution: Marc T. Facciotti (own work)

 

Light Independent Reactions and Carbon Fixation

A short introduction

The general principle of carbon fixation is that some cells under certain conditions can take inorganic carbon, CO₂ (also referred to as mineralized carbon), and reduce it to a usable cellular form. Most of us know that green plants can take up CO₂ and produce O₂ in a process known as photosynthesis. We have already discussed photophosphorylation, the ability of a cell to transfer light energy onto chemicals and ultimately to produce the energy carriers ATP and NADPH in a process known as the light reactions. In photosynthesis, the plant cells use the ATP and NADPH formed during photophosphorylation to reduce CO₂ to sugar, (as we will see, specifically G3P) in what we call the dark reactions. While we appreciate that this process happens in green plants, photosynthesis had its evolutionary origins in the bacterial world. In this module we will go over the general reactions of the Calvin Cycle, a reductive pathway that incorporates CO₂ into cellular material.

In photosynthetic bacteria, such as Cyanobacteria and purple non-sulfur bacteria, as well plants, the energy (ATP) and reducing power (NADPH) - a term used to describe electron carriers in their reduced state - obtained from photophosphorylation, is coupled to "Carbon Fixation", incorporating inorganic carbon (CO₂) into organic molecules; initially as glyceraldehyde-3-phosphate (G3P) and eventually into glucose. We refer to organisms that can get all of their required carbon from an inorganic source (CO₂) as autotrophs, while we refer to those organisms that require organic forms of carbon, such as glucose or amino acids, as heterotrophs. The biological pathway that leads to carbon fixation is called the Calvin Cycle and is a reductive pathway (consumes energy/uses electrons) which leads to the reduction of CO₂ to G3P.

 

The Calvin Cycle: the reduction of CO₂ to Glyceraldehyde 3-Phosphate

Figure 1. Light reactions harness energy from the sun to produce chemical bonds, ATP, and NADPH. These energy-carrying molecules are made in the stroma where carbon fixation takes place.

In plant cells, the Calvin cycle is located in the chloroplasts. While the process is similar in bacteria, there are no specific organelles that house the Calvin Cycle and the reactions occur in the cytoplasm around a complex membrane system derived from the plasma membrane. This intracellular membrane system can be quite complex and highly regulated. There is strong evidence that supports the hypothesis that the origin of chloroplasts from a symbiosis between cyanobacteria and early plant cells.

Stage 1: Carbon Fixation

In the stroma of plant chloroplasts, besides CO₂, two other components are present to start the light-independent reactions: an enzyme called ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and three molecules of ribulose bisphosphate (RuBP), as shown in the figure below. Ribulose-1,5-bisphosphate (RuBP) is composed of five carbon atoms and includes two phosphates.

Figure 2. The Calvin cycle has three stages. In stage 1, the enzyme RuBisCO incorporates carbon dioxide into an organic molecule, 3-PGA. In stage 2, the organic molecule is reduced using electrons supplied by NADPH. In stage 3, RuBP, the molecule that starts the cycle, is regenerated so that the cycle can continue. Only one carbon dioxide molecule is incorporated at a time, so it must complete the cycle three times to produce a single three-carbon GA3P molecule, and six times to produce a six-carbon glucose molecule.

RuBisCO catalyzes a reaction between CO₂ and RuBP. For each CO₂ molecule that reacts with one RuBP, two molecules of another compound (3-PGA) form. PGA has three carbons and one phosphate. Each turn of the cycle involves only one RuBP and one carbon dioxide and forms two molecules of 3-PGA. The number of carbon atoms remains the same, as the atoms move to form new bonds during the reactions (3 atoms from 3CO₂ + 15 atoms from 3RuBP = 18 atoms in 3 atoms of 3-PGA). We call this process carbon fixation, because CO₂ is “fixed” from an inorganic form into an organic molecule.

Stage 2: Reduction

ATP and NADPH are used to convert the six molecules of 3-PGA into six molecules of a chemical called glyceraldehyde 3-phosphate (G3P) - a carbon compound also found in glycolysis. The process uses six molecules of both ATP and NADPH. The exergonic process of ATP hydrolysis is in effect driving the endergonic redox reactions, creating ADP and NADP⁺. Both "spent" molecules (ADP and NADP⁺) return to the nearby light-dependent reactions to be recycled back into ATP and NADPH.

Stage 3: Regeneration

Interestingly, at this point, only one of the G3P molecules leaves the Calvin cycle to contribute to the formation of other compounds needed by the organism. In plants, because the G3P exported from the Calvin cycle has three carbon atoms, it takes three “turns” of the Calvin cycle to fix enough net carbon to export one G3P. But each turn makes two G3Ps, thus three turns make six G3Ps. One is exported while the remaining five G3P molecules remain in the cycle and are used to regenerate RuBP, which enables the system to prepare for more CO₂ to be fixed. These regeneration reactions use three more molecules of ATP.

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Possible NB Discussion Point

Have you ever heard anyone accidentally refer to the Amazon rainforest as the "lungs of the Earth"? In reality, the majority of our planet's oxygen is produced by marine organisms, such as microscopic phytoplankton -- which, by the way, also take up appreciable amounts of carbon dioxide from the environment. The family of phytoplankton include organisms like cyanobacteria and diatoms (a visually stunning type of algae -- look it up!) that are able to survive and aggregate close to the water's surface, where sun exposure is higher. Try to approach phytoplankton from a BIS 2A lens... What biochemical processes had to happen in order for these phytoplankton to produce oxygen? What exactly are the phytoplankton doing with the carbon dioxide they take up from the atmosphere? What large-scale global effects would you expect if phytoplankton health were to be severely compromised?

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Additional Links of Interest

Khan Academy Links

• Calvin Cycle

Chemwiki links

• Calvin Cycle

YouTube Videos

• 3D animation of photosynthesis in plants

• Calvin Cycle