Lecture 10: Photophosphoryaltion/ATP production and Carbon Fixation

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 possible radiation frequencies. When solar radiation reaches Earth, a fraction of this energy interacts with and may be transferred to the matter on the planet. This energy transfer results in a wide variety of different phenomena, from influencing weather patterns to driving a myriad of 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 couple of 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.

The distance between peaks in a wave is referred to as the wavelength and is abbreviated 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 drawn 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*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 has 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 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 different mechanisms, some of which you may learn about in upper division courses in cellular physiology or biophysical chemistry. In BIS2A, we are mostly concerned with the interaction of light and biological pigments. These interactions can initiate a variety of light-dependent biological processes that can be grossly grouped into two functional categories: cellular signaling and energy harvesting. Signaling interactions are largely responsible for perceiving 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. By contrast, 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. Photosynthesis, which we will learn more about soon, is one example of an energy harvesting interaction.  

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 We Discuss in BIS2A

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".

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. In other words, the excited state can be involved in redox reactions.

As the excited electron decays back to its lower energy state, the energy can be transferred 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 is sufficient to allow the reaction center to donate the electron in a redox reaction to a second molecule. This initiates the electron transport reactions. The result is an oxidized reaction center that must now be reduced in order to start the process again. How this happens is the basis of electron flow in photophosphorylation and will be described in detail below. 

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 redox reactions. These carriers are the same carriers found in respiration. If the change in reduction potential from the various redox 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 P480 reaction center is not necessarily the same electron that eventually finds its way back to reduce the oxidized P840. 

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 are removed from the photosystem and redox 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 be used to reduce the oxidized form of bacteriochlorophyll. The second requirement is that, when bacteriochlorophyll becomes oxidized and the electron is ejected, it must reduce a carrier that has a greater reduction potential than NADP/NADPH (see the electron tower). In this case, electrons can flow from energized bacteriochlorophyll to NADP forming NADPH and oxidized bacteriochlorophyll. Electrons are lost from the system and end up on NADPH; to complete the circuit, bacteriochlorophyll_ox is reduced by an external electron donor such as H₂S or elemental S⁰.

Generation of NADPH and ATP

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

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 on the basis of 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; a number of antenna proteins to which the chlorophyll molecules are bound surround the reaction center in which the photochemistry takes place. Each photosystem is serviced by the light-harvesting complex, which passes energy from sunlight to the reaction center; it consists of multiple antenna proteins that contain a mixture of 300–400 chlorophyll a and b molecules as well as 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 light energy has now been captured by biological molecules but is not yet stored in any useful form. The captured energy is transferred from chlorophyll to chlorophyll until, eventually (after about a millionth of a second), it is delivered to the reaction center. Up to this point, only energy has been transferred between molecules, not electrons.

The reaction center contains a pair of chlorophyll a molecules with a special property. Those two chlorophylls can undergo oxidation upon excitation; they can actually 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 is transferred into an excited electron. All of the subsequent steps involve getting 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 electron transport chain

PSII and PSI are two major components of the photosynthetic electron transport chain, which also includes the cytochrome complex. The cytochrome complex, an enzyme composed of two protein complexes, transfers the electrons from the carrier molecule plastoquinone (Pq) to the protein plastocyanin (Pc), thus enabling both the transfer of protons across the thylakoid membrane and the transfer of electrons from PSII to PSI.  

The reaction center of PSII (called P680) delivers its high-energy electrons, one at a time, to a primary electron acceptor, and through the electron transport chain (Pq to cytochrome complex to plastocyanine) to PSI. P680’s missing electron is replaced by extracting an electron from water; thus, water is split and PSII is re-reduced after every photoactivation step. Splitting one H₂O molecule releases two electrons, two hydrogen atoms, and one atom of oxygen. Splitting two molecules of water is required to form one molecule of diatomic O₂ gas. In plants, about ten percent of that oxygen is used by mitochondria in the leaf to support oxidative phosphorylation. The remainder escapes to the atmosphere where it is used by aerobic organisms to support respiration.

As electrons move through the proteins that reside between PSII and PSI, they take part in exergonic redox transfers. The free energy associated with the exergonic redox reaction is coupled to the endergonic transport of protons from the stromal side of the membrane to the thylakoid lumen. Those hydrogen ions, plus the ones produced by splitting water, accumulate in the thylakoid lumen and create a proton motive force that will be used to 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 redox 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 is transferred to the PSI reaction center (called P700). P700 is oxidized and sends an electron through several intermediate redox steps to NADP⁺ to form NADPH. Thus, PSII captures the energy in light and couples its transfer via redox 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 redox 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 exactly match the chloroplast’s constantly changing energy needs.

Figure 8. A diagram depicting the flow of electrons and the redox potentials of their carriers in oxygenic photosynthetic systems expressing both photosystem I and photosystem II.  
Attribution: Marc T. Facciotti (own work)

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 are aware 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 are called 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", the incorporation of inorganic carbon (CO₂) into organic molecules; initially as glyceraldehyde-3-phosphate (G3P) and eventually into glucose. Organisms that can obtain all of their required carbon from an inorganic source (CO₂) are referred to as autotrophs, while those organisms that require organic forms of carbon, such as glucose or amino acids, are referred to 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.