Light-Driven Electron Flow

Thylakoid membranes have two different kinds of photosystems, each with its own type of photochemical reaction center and a set of antenna molecules. The two systems have distinct and complementary functions. Photosystem I has a reaction center designated P700 and a high ratio of chlorophyll a to chlorophyll b. Photosystem II, with its reaction center P680, contains roughly equal amounts of chlorophyll a and b and may also contain a third type, chlorophyll c. The thylakoid membranes of a single spinach chloroplast have many hundreds of each kind of photosystem. All O2-evolving photosynthetic cells-those of higher plants, algae, and cyanobacteria-contain both photosystems I and II; all other species of photosynthetic bacteria, which do not evolve O2, contain only photosystem I.

It is between photosystems I and II that light-driven electron flow occurs, producing NADPH and a transmembrane proton gradient.

Light Absorption by Photosystem II Initiates Charge Separation

How can light energy captured by chloroplasts induce electrons to flow energetically "uphill"? Excitation briefly creates a chemical species of very low standard reduction potential-an excellent electron donor. Excited P680, designated P680*, within picoseconds transfers an electron to pheophytin (a chlorophyll-like accessory pigment lacking Mg2+), giving it a negative charge (designated Ph- ) (Fig. 18-41a). With the loss of its electron, P680* is transformed into a radical cation, designated P680+. Thus excitation, in creating Ph- and P680* , causes charge separation. Ph- very rapidly passes its extra electron to a protein-bound plastoquinone, QA, which in turn passes its electron to another, more loosely bound quinone, QB (Fig. 18-42; see also Fig. 1844). When QB has acquired two electrons in two such transfers from QA and two protons from the solvent water, it is in its fully reduced quinol form, QBH2. This molecule dissociates from its protein and diffuses away from the photochemical reaction center, carrying in its chemical bonds some of the energy of the photons that originally excited P680. The overall reaction initiated by light in photosystem II is therefore

4 P680 + 4H+ + 2QB + light (4 photons) 4 P680+ + 2QBH2 ........... (18-4)

Figure 18-41 Photochemical events following excitation of photosystems by light absorption. (The steps shown here are equivalent to steps 4 and 5 in Fig. 18-40.) (a) Photosystem II (PSII). Z represents a Tyr residue in the D1 protein of PSII; Ph, pheophytin. (b) Photosystem I (PSI). A0 is a chlorophyll molecule near the reaction center of PSI; it accepts an electron from P700 to become the powerful reducing agent A0-, . PC, plastocyanin.

Figure 18-42 (a) Plastoquinone (QA). (b) The herbicide DCMU (3-(3,4-dichlorophenyl)-1, 1-dimethylurea ) , which displaces QB from its binding site in photosystem II and blocks electron transfer from photosystem II to photosystem I. (c) The role of QA and QB in transferring electrons away from photosystem II. QBH2 carries some of the energy of light absorbed by PSII.

Eventually, the electrons in QBH2 are transferred through a chain of membrane-bound carriers to NADP+, reducing it to NADPH and releasing H+ (as described later). The potent herbicide DCMU (Fig. 1842) competes with QB for the QB binding site in photosystem II, thus blocking photosynthetic electron transfer (Table 18-4).

In the meantime, P680+ must acquire an electron to return to its ground state in preparation for the capture of another photon of energy (Fig. 18-41a). In principle, the required electron might come from any number of organic or inorganic compounds. Photosynthetic bacteria can use a variety of electron donors for this purpose-acetate, succinate, malate, or sulfide-depending on what is available in a particular ecological niche. About 3 billion years ago, evolution of primitive photosynthetic bacteria (the progenitors of the modern cyanobacteria) produced a photosystem capable of taking electrons from a donor that is always available-water. In this process two water molecules are split, yielding four electrons, four protons, and molecular oxygen: 2H2O 4H+ + 4e- + O2. A single photon of visible light does not possess enough energy to break the bonds in water; four photons are required in this photolytic cleavage reaction.

The four electrons abstracted from water do not pass directly to P680+, which can only accept one electron at a time. Instead, a remarkable molecular device, the water-splitting complex, passes four electrons one at a time to P680+. The immediate electron donor to P680+ is a Tyr residue (often represented by the symbol Z) in protein D1 of the photosystem II reaction center:

This Tyr residue regains its missing electron by oxidizing a cluster of four manganese ions in the water-splitting complex. With each singleelectron transfer, this Mn cluster becomes more oxidized; four singleelectron transfers, each corresponding to the absorption of one photon, produce a charge of +4 on the Mn complex (Fig. 18-43):

In this state, the Mn complex can take four electrons from a pair of water molecules, releasing 4H+ and 02:

[Mn complex]4+ + 2 H2O [Mn complex]0 + 4H+ + O2

The sum of Equations 18-4 through 18-7 is

The water-splitting activity is an integral part of the photosystem II reaction center, and it has proved exceptionally difficult to purify. The detailed structure of the Mn cluster is not yet known. Manganese can exist in stable oxidation states from +2 to +7, so a cluster of four Mn ions can certainly donate or accept four electrons; the chemical details of this process, however, remain to be clarified.

Figure 18-43 The four-step process that produces a four-electron oxidizing agent, believed to be a complex of several Mn ions, in the water-splitting complex of photosystem II. The sequential absorption of four photons, each causing the loss of one electron from the Mn center, produces an oxidizing agent that can take four electrons from two molecules of water, producing O2. The electrons lost from the Mn center pass one at a time to a Tyr residue (Z+) in a reaction-center protein.

Light Absorption by Photosystem I Creates a Powerful fteducing Agent

The photochemical events that follow excitation of photosystem I (P700) are formally similar to those in photosystem II (following the general scheme of Figure 18-40). Light is first captured by any one of about 200 chlorophyll a and b molecules, or additional accessory pigments, that serve as antennae, and the absorbed energy moves to P700 by resonance energy transfer. The excited reaction center P700* loses an electron to an acceptor, Ao (believed to be a special form of chlorophyll, functionally analogous to the pheophytin of photosystem II), creating Ao and P700+ (Fig. 18-41b); again, excitation has resulted in charge separation at the photochemical reaction center. P700+ is a strong oxidizing agent, which quickly acquires an electron from plastocyanin, a soluble Cu-containing electron transfer protein.

A0- is an exceptionally strong reducing agent, which passes its electron through a chain of carriers that leads to NADP+ (Fig. 18-44). First, phylloquinone (A1) accepts an electron from A0 and passes it on to an iron-sulfur protein. From here, the electron moves to ferredoxin (Fd), another iron-sulfur protein loosely associated with the thylakoid membrane. Spinach ferredoxin (Mr 10,700), which has been isolated and crystallized, contains a 2Fe-2S center (Fig. 18-5). The Fe atoms of ferredoxin transfer electrons via one-electron Fe2+ to Fe3+ valence changes.

Figure 18-44 The integration of photosystems I and II. This "Z scheme" shows the pathway of electron transfer from H2O (lower left) to NADP+ (upper right) in noncyclic photosynthesis. The position on the vertical scale of each electron carrier reflects its standard reduction potential. To raise the energy of electrons derived from H2O to the energy level required to reduce NADP+ to NADPH, each electron must be "lifted" twice (heavy arrows) by photons absorbed in photosystems I and II. One photon is required per electron boosted in each photosystem. After each excitation, the high-energy electrons flow "downhill" via the carrier chains shown. Protons move across the thylakoid membrane during the water-splitting reaction and during electron transfer through the cytochrome bf complex, producing the proton gradient that is central to ATP formation. The dashed arrow is the path of cyclic electron transfer, in which only photosystem I is involved; electrons return via the cyclic pathway to photosystem I, instead of reducing NADP+ to NADPH. Ph, pheophytin; QA, plastoquinone; QR, a second quinone; PC, plastocyanin; A0 electron acceptor chlorophyll; A1, phylloquinone; Fd, ferredoxin; FP, ferredoxin-NADP+ oxidoreductase.

The fourth electron carrier in the chain is a flavoprotein called ferredoxin-NADP+ oxidoreductase. It transfers electrons from reduced ferredoxin (Fd2+red) to NADP+, reducing the latter to NADPH:

Photosystems I and II Cooperate to Carry Electrons from H2O to NADP+

Early studies by Robert Emerson established that maximum rates of photosynthesis, measured as O2 evolution, required light of at least two wavelengths, now known to excite photosystems I and II. The two photosystems must function together in the O2-evolving light reactions of photosynthesis. The diagram in Figure 18-44, often called the Z scheme because of its overall form, outlines the pathway of electron flow between the two photosystems as well as the energy relationships in the light reactions.

When photons are absorbed by photosystem I, electrons are expelled from the reaction center and flow down a chain of electron carriers to NADP+ to reduce it to NADPH. P700+, transiently electrondeficient, accepts an electron expelled by illumination of photosystem II, which arrives via a second connecting chain of electron carriers. This leaves an "electron hole" in photosystem II, which is filled by electrons from H2O. We have described how water is split to yield: (1) electrons, which are donated to the electron-deficient photosystem II; (2) H+ ions (protons), which are released inside the thylakoid lumen; and (3) O2, which is released into the gas phase. The Z scheme thus describes the complete route by which electrons flow from H2O to NADP+ according to the equation

2H2O + 2 NADP+ + 8 photons O2 + 2NADPH + 2H+

For each electron transferred from H2O to NADP+, two photons are absorbed, one by each photosystem. To form one molecule of O2, which requires transfer of four electrons from two H2O to two NADP+, a total of eight photons must be absorbed, four by each photosystem.

The Cytochrome bf Complex Links Photosystems II and I

Electrons stored in QBH2 as a result of the excitation of P680 in photosystem II (Fig. 18-42c) are carried to P700 of photosystem I via an assembly of several integral membrane proteins known as the cytochrome bf complex and the soluble protein plastocyanin (Fig. 18-44). The purified cytochrome bf complex contains a b-type cytochrome with two heme groups (cytochrome b563), an iron-sulfur protein (Mr 20,000), and cytochrome f (named for the Latin frons, meaning "leaf"), also called cytochrome c552?Electrons flow through the cytochrome bf complex from QBH2 to cytochrome f; the detailed path is uncertain. Cytochrome f passes its electron to plastocyanin, the donor for P700 reduction (Figs. 18-41b, 18-44).

The cytochrome bf complex of plants is remarkably similar to the cytochrome bc1 complex (Complex III) of the mitochondrial electron transfer chain, and it carries out a similar function. Both complexes convey electrons from a reduced quinone, a mobile, lipid-soluble carrier of two electrons (UQ in mitochondria, QB in chloroplasts), to a watersoluble protein that carries one electron (cytochrome c in mitochondria, plastocyanin in chloroplasts). As in mitochondria, the function of this complex involves a Q cycle (see Fig. 18-10) in which electrons pass from QBH2 to cytochrome b one at a time. As in the mitochondrial Complex III, this cycle results in the pumping of protons across the membrane; in chloroplasts, the direction of proton movement is from the stromal compartment to the thylakoid lumen. The result is the production of a proton gradient across the thylakoid membrane as electrons pass from photosystem II to photosystem I (Fig. 18-45). Because the volume of the flattened thylakoid lumen is small, the influx of a small number of protons has a relatively large effect on lumenal pH. The measured difference in pH between the stroma (pH 8) and the thylakoid lumen (pH 4.5) represents a 3,000-fold difference in proton concentration-a powerful driving force for ATP synthesis.

Figure 18-45 Proton and electron circuits in chloroplast thylakoids. Electrons (blue arrows) move from H2O through photosystem II, the intermediate chain of carriers, photosystem I, and finally to NADP+. Protons (red arrows) are pumped into the thylakoid lumen by the flow of electrons through the chain of carriers between photosystem II and photosystem I, and reenter the stroma through proton channels formed by the F0 portion of the ATP synthase, designated CF0 in the chloroplast enzyme. The Fl subunit (CF1) catalyzes synthesis of ATP.