by

Govindjee and Rajni Govindjee

University of Illinois at Urbana-Champaign

Copyright © by Govindjee and Rajni Govindjee 2000

Whenever molecules gain or lose electrons energy is involved. The Z-scheme is an energy diagram for electron transfer in the "light reactions" of plant photosynthesis. It applies equally well to photosynthesis by algae and cyanobacteria. The vertical energy scale shows each molecule’s ability to transfer an electron to (i.e., to reduce) the next one from left to right. The ones at the top transfer electrons easily to the ones below them as it is a "downhill" reaction, energy-wise. However, for electron transfer from those at the bottom to those above them it is an "uphill" reaction and requires input of outside energy. The Z scheme shows the pathway of electron transfer from water to NADP⁺. Using this pathway, plants transform light energy into "electrical" energy (electron flow) and hence into chemical energy as reduced NADPH and ATP. Later in the "dark reactions" of photosynthesis, that chemical energy is further transformed into the chemical bonds in sugar molecules. In the complete process, carbon dioxide is joined into the sugar molecules and oxygen is released. Although it is not a structural diagram, the Z-scheme does give the sequence of electron flow (oxidation and reduction) with an energy perspective. The source of electrons is water (H₂O). The large vertical red arrows represent excitation of reaction center chlorophyll molecules (by light energy) and the black arrows represent electron flow, which is downhill energy-wise. This particular version of the "Z-scheme" was developed for the novel, THE MUSIC OF SUNLIGHT by Sunlight Books in collaboration with the authors of this article.

Mn is the manganese center, a complex containing 4 manganese atoms, which splits two water molecules at a time into 4 protons (4H⁺), 4 electrons (4e^-), and 2 oxygen atoms, as an oxygen molecule (O₂). Tyr is a special tyrosine molecule, also sometimes referred to as Yz or simply as Z, which shuttles electrons to the "reaction center" of photosystem II (PSII). Chl P680 is the reaction center pair of chlorophyll a molecules of PSII. Excited Chl P680* has reached this state by absorbing a photon of light energy. Pheo is a pheophytin molecule, which is a chlorophyll with its central magnesium ion (Mg⁺⁺) having been replaced by two hydrogens. It is the primary electron acceptor of PSII, whereas P680 is the primary electron donor. Q_A is a plastoquinone molecule, which is tightly-bound and immovable. It is known in some circles as the primary stable electron acceptor of PSII, and it accepts and transfers one electron at a time. Q_B is a loosely bound plastoquinone molecule, which accepts two electrons and then takes on two protons, before it detaches and becomes mobile and called PQ. PQ is the detached plastoquinone molecule, which is mobile within the hydrophobic core of the thylakoid membrane. FeS is the Rieske iron-sulfur protein. Cyt f is cytochrome f. Cyt b_6L and Cyt b_6H are two cytochrome b₆ molecules (of lower and higher energy). PC is plastocyanin, a highly mobile copper protein. Chl P700 and Excited Chl P700* are respectively the ground energy state and the excited energy state of the chlorophyll molecule of the "reaction center" of photosystem I. A_O is a special chlorophyll a molecule that is the primary electron acceptor of PSI, whereas P700 is the primary electron donor of PSI. A₁ is a phylloquinone (vitamin K) molecule. F_X, F_A, and F_B are three separate immobile iron-sulfur protein centers. FD is ferredoxin, a somewhat mobile iron-sulfur protein. FNR is the enzyme ferredoxin-NADP oxidoreductase, which contains the active group, called FAD (flavin adenine dinucleotide). NADP⁺ is the oxidized form of nicotinamide adenine dinucleotide phosphate. NADPH is its reduced form. .

The Sun is the source of almost all energy that runs life on Earth. Each minute our Sun converts 120 million tons of its mass into electromagnetic radiation and dumps it out into space. Only one billionth of that reaches Earth. It takes only 8 minutes for this radiation to travel 93 million miles to us. The visible portion of this electromagnetic radiation (VIBGYOR: violet, indigo, blue, green, yellow, orange and red - the colors in our rainbow) is captured by plants, algae and cyanobacteria. Plants are green because chlorophyll, the key pigment that captures light, absorbs blue and red efficiently, but transmits most of the green light for our eyes to see. Photosynthetic organisms then use the absorbed light energy to make food (that we need to eat) and oxygen (that we need to breathe). In addition, past photosynthesis provided the "fossil fuel" we need to drive our cars.

The process of photosynthesis in oxygen-producing organisms involves the massive conversion of sunlight into sugars and oxygen, using carbon dioxide, water and some minerals as ingredients. Almost all life-forms (except those in the hot vents of the ocean floor) depend on photosynthesis. Plants make leaves, roots, stems and fruits that herbivores (such as caterpillars) eat. Carnivores eat herbivores and omnivores eat both. There is this "web of life". Photosynthesis is the basis of this food web that links almost all living beings on Earth. In the oceans small fish eat the phytoplankton (algae), the bigger fish eat the small fish and the "web of life" goes on. Plants capture only one thousandth of the sunlight that falls on the Earth. Yet, without this process all life would come to a halt. In 1969, E. Rabinowitch and Govindjee wrote: "A living organism is like a running clock. If it is not wound up, it will sooner or later run out of energy and stop. If the clock of life on earth would be left to run down without rewinding, it would take less than one hundred years for all life on the planet to approach its end. First green plants would die from starvation. Humans and other animals who feed on plants would follow. And finally, bacteria and fungi feeding on dead animal and plant tissues would exhaust their food and die too."

Photosynthesis also links plants and animals through the air, since oxygen released by plants is used for respiration by both plants and animals. Respiration, in turn, releases carbon dioxide that the plants use to perform photosynthesis. Humans are putting extra carbon dioxide into the air by burning fossil fuels. This increased CO₂ is leading to higher global temperatures. Recently it has been stated that the temperatures in the Himalayan mountain range may have been much higher in the last 100 years than ever before. The increased carbon dioxide and increased temperatures have serious consequences in our future lives, those of our children and of our grandchildren. We should stop and think and try to understand the photosynthetic process.

Photosynthesis occurs in green plants just as it does in algae and in certain bacteria. In plants, it is the leaves that do it, but only those leaf cells that contain the organelles, called chloroplasts, can perform this process. Inside each chloroplast are "thylakoid membranes", which form connected flattened sacs (thylakoid means sac-like in Greek). These thylakoids are arranged in stacks of sacs called grana. (Imagine stacks of hollow chapitis or pita bread.) The water-filled space outside the sacs is called the stroma matrix and the water-filled space within them is called the lumen.

In plants (as well as algae and cyanobacteria), photosynthesis has two major phases:

1. The Light Reactions comprise the light-dependent phase, which produces the reducing power ("reducing", as in "oxidation and reduction"), ATP (adenosine triphosphate – the energy currency of life), and oxygen (O₂). This all takes place in and around the thylakoid membranes.
2. The Dark Reactions are not directly dependent on the presence of light. Here the reducing power of NADPH and the energy of ATP (both generated by the light reactions) are used to convert, or "fix" CO₂ into sugars. These reactions occur in the stroma matrix and are called the Calvin-Benson cycle or C3 cycle. They are not shown in the Z-scheme diagram. The pathway for ATP production is also not shown in the diagram.

It is simply because the diagram, when first drawn, was in the form of the letter "Z". Actually, you can see such a diagram in the older literature. (See e.g., Govindjee & R. Govindjee (1975) in: Bioenergetics of Photosynthesis, Academic Press, P. 27: and Demeter, S. and Govindjee (1989) Physiologia Plantarum, Volume 75, Pages 121-130. Currently, it is being drawn to emphasize the energy levels of the components. Thus, it has been turned 90 degrees counterclockwise.

Excitation of Reaction Centers Photosynthesis starts with the simultaneous excitation of pairs of special reaction center chlorophyll(a) molecules, labeled as P680 (in photosystem II, or PSII) and P700 (in photosystem I, or PSI). [See the two red vertical arrows in the diagram.] The excitation energy comes either from directly absorbed light or (most often) by energy transfer from adjacent pigment molecules in protein complexes called antennas. These "antenna" pigment molecules (chlorophylls and carotenoids) absorb light energy and then transmit it by inductive resonance from one molecule to the next, finally to the reaction center. Excitation is over within a few femtoseconds (10^-15 s). [A second has as many femtoseconds in it as 31 million years has seconds.]

The Electron Transfer Steps A molecule with a plus (+) charge has one less electron than its counterpart and is said to be the oxidized species, as it has lost one electron. The species that has an added electron is called the reduced species. Reduction means the addition of electrons or of hydrogen atoms {One hydrogen atom is a combination of one proton (H⁺) and one electron (e^-)}, and oxidation means removal of either H or e^-. This oxidation and reduction process is what drives the activity in the Z-scheme sequence. Molecules higher on the diagram are able to reduce (pass an electron to) the next molecule lower down on the energy scale.

The recovery (reduction) of P680⁺ to P680 and of P700⁺ to P700 happens almost simultaneously. P700⁺ recovers to P700 by receiving an electron that was passed down from reduced Pheo to Q_A (which is bound to the reaction center protein complex), then to Q_B (another bound plastoquinone molecule). When Q_B has accepted two electrons from Q_A, it also takes on two protons from the stroma. Then it detaches from its protein binding site and diffuses through the hydrophobic core of the thylakoid membrane to the Cyt bf complex (see below), where the electrons are passed on to an iron-sulfur protein (FeS, the Rieske protein) and then to a mobile copper-protein (PC, or plastocyanin) which finally carries a single electron to the oxidized P700⁺. Thus the electron is passed in "bucket brigade" manner through the "intersystem chain of electron (or H-atom) carriers".

There is a protein complex called the Cyt bf complex (shown as a grey rectangle on this diagram) which contains FeS, Cytochrome f, and two cytochrome b₆ molecules. The "bottleneck", or the slowest step of the entire sequence, is the passage of the reduced PQ molecule to the Cyt bf complex and PQ’s oxidation by FeS. This takes several milliseconds (10^-3 s). Cytochrome b₆ is active in the Q-cycle (see below).

In PSI the electron on A_O^- is passed ultimately to NADP⁺ via several other intermediates: A₁, a phylloquinone (vitamin K); F_X, F_A, and F_B which are immobile (bound) iron-sulfur proteins; ferredoxin, which is a somewhat mobile iron-sulfur protein molecule; and the enzyme ferredoxin-NADP reductase (FNR) which is actually an oxido-reductase and whose active group is FAD (short for flavin adenine dinucleotide).

The missing electron on P680⁺ is recovered, ultimately, from water molecules on the left bottom of the diagram via an amino acid tyrosine (a specific one in a protein of PSII, also referred to as Yz in the literature) and a tetra-nuclear manganese (Mn) cluster. These reactions also require a few milliseconds.

A total of 8 quanta (photons) of light (4 in PSII and 4 in PSI), are required to transfer 4 electrons from 2 molecules of water to 2 molecules of NADP⁺. This produces 2 molecules of NADPH and 1 molecule of O₂. This is the oxygen that both plants and animals need for respiration and life.

The left side of the diagram shows an energy scale in terms of oxidation-reduction potential (E_m) at pH 7. {At pH 7 the standard hydrogen electrode has an E_m of –0.4 volts.} Intermediates that are higher up in the diagram have a lower (more negative) E_m and can easily reduce (i.e., add an electron to) any intermediate below them in the diagram by a downhill energy-wise process. This occurs in electron transfer:

1. from Pheo^- to P700⁺ (see middle of diagram)
2. from A_O^- to NADP⁺ (see top right end of diagram)
3. from H₂O to P680⁺ (see lower left of diagram)

Energy is needed to transfer electrons from P680 to Pheo and from P700 to A_O, and this is where "light" energy is consumed.

The light reactions provide not only the reducing power in NADPH but also the energy of ATP, both essential for producing sugars from CO₂. ATP is produced through an enzyme called ATP synthase, using ADP (adenosine diphosphate), inorganic phosphate (P_i) and energy from a proton motive force (pmf) across the thylakoid membrane. This pmf is composed of two components:an electrical potential across the thylakoid membrane and a proton gradient across the thylakoid membrane. The proton gradient comes from the storing up of protons (hydrogen ions) inside the lumen, giving a pH of 6 inside the lumen and pH of 8 outside, in the stroma. Then, basically, protons escaping out from the thylakoid lumen through a central core of the enzyme ATP synthase (embedded in the membrane) cause conformational (rotational) changes in the enzyme, which catalyzes the phosphorylation of ADP and the release of ATP on the stromal side.

Protons (hydrogen ions) are concentrated into the lumen in several ways:

1. Oxidation of water not only releases O₂ and "sends" electrons to P680, but it also releases protons (H⁺) into the lumen.
2. When Q_B is reduced in PSII, it not only receives two electrons from Q_A but it also picks up two protons from the stroma matrix and becomes PQH₂. It is able to "carry" both electrons and protons (hydrogens). It is a H-atom carrier. At the Cyt bf complex it is then oxidized, but FeS and Cyt b₆ can only accept electrons (not protons). So the two protons are released into the lumen.
3. The Q-cycle of the Cyt bf complex is great because it provides extra protons into the lumen. Here two electrons travel through the two hemes of cytochrome b₆ and then reduce PQ on the stroma side of the membrane. The reduced PQ takes on two protons from the stroma, becoming PQH₂, which migrates to the lumen side of the Cyt bf complex where it is again oxidized, releasing two more protons into the lumen. Thus the Q-cycle allows formation of more ATP..
4. When NADP⁺ is reduced by two electrons, it also picks up one proton, in effect removing it from the stroma and further increasing the gradient across the membrane.

The Z-scheme owes its origin to several investigators. First it was Robert Emerson and his co-workers, including the authors (1956-1964) at the University of Illinois (at Urbana-Champaign) who discovered and embellished the "Enhancement effect" in oxygen evolution, which occurred when light absorbed in one photosystem (currently called PSI) was added to light absorbed in another photosystem (currently called PSII). Experiments using chloroplasts, and those using a mass spectrometer, absorption spectrometer, a fluorometer and electron spin resonance spectrometer were crucial to the establishment of the concepts. These suggested the existence of "two light reactions and two photosystems". It was Bessel Kok and co-workers (1959-1960) at Baltimore, Maryland, and Lou NM Duysens, J. Amesz and co-workers (1959-1961) in Leiden, The Netherlands, who discovered the crucial antagonistic effect of light absorbed in PSI and PSII on the oxidation-reduction state of P700 and of cytochrome f (Cyt f, the electron carrier in the middle of the intersystem chain of intermediates), respectively. Duysens’s experiments established the "series" nature of the present scheme. Light captured by PSI, in the "Z"-scheme, leads to oxidation of Cyt f (i.e., takes an electron away from it and places it on, say, "NADP⁺), whereas when light is captured by PSII, oxidized Cyt f is reduced by an electron coming from PSII. The theoretical concepts of Robin Hill and Fay Bendall (1960) (who essentially gave us the Z-scheme), the earlier schemes in books by Eugene Rabinowitch (1945-1956), and the detailed work of Horst Witt and co-workers (1961) in Berlin, Germany, played important and crucial roles in the origin of the "Z-scheme". The final evidence of its validity came from state-of-the-art detailed biophysics, biochemical, molecular biology, and genetic research in about 20 laboratories around the world. This is not to say that mutants and organisms may not be found in the future, that use alternate means to transfer electron from water to NADPH⁺.