Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

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● The Z scheme of photosynthesis. (a) The Z scheme is a diagrammatic representation of photosynthetic electron flow from H2O to NADP . The energy relationships can be derived from the scale beside the Z diagram, with lower standard potentials and hence greater energy as you go from bottom to top. Energy input as light is indicated by two broad arrows, one photon appearing in P680 and the other in P700. P680* and P700* represent photoexcited states. Electron loss from P680* and P700* creates P680 and P700 . The representative components of the three supramolecular complexes (PSI, PSII, and the cytochrome b6/cytochrome f complex) are in shaded boxes enclosed by solid black lines. Proton translocations that establish the proton-motive force driving ATP synthesis are illustrated as well. (b) Figure showing the functional relationships among PSII, the cytochrome b/cytochrome f complex, PSI, and the photosynthetic CF1CF0 ATP synthase within the thylakoid membrane. Note that e acceptors QA (for PSII) and A1 (for PSI) are at the stromal side of the thylakoid membrane, whereas the e donors to P680 and P700 are situated at the lumenal side of the membrane. The consequence is charge

separation ( stroma, lumen) across the membrane. Also note that protons are translocated into the thylakoid lumen, giving rise to a chemiosmotic gradient that is the driving force

for ATP synthesis by CF1CF0 ATP synthase.

722 Chapter 22 ● Photosynthesis

O H

Plastohydroquinone A

● The structures of plastoquinone and its reduced form, plastohydroquinone (or plastoquinol). The oxidation of the hydroquinone releases 2 H as well as 2 e . The form shown (plastoquinone A) has nine isoprene units and is the most abundant plastoquinone in plants and algae. Other plastoquinones have different numbers of isoprene units and may vary in the substitutions on the quinone ring.

Electron Transfer Within the

Cytochrome b6/Cytochrome f Complex

The cytochrome b6/cytochrome f or plastoquinol:plastocyanin oxidoreductase is a large (210 kD) multimeric protein possessing 22 to 24 transmembrane-helices. It includes the two heme-containing electron transfer proteins for which it is named as well as iron–sulfur clusters (Chapter 21), which also participate in electron transport. The purpose of this complex is to mediate the transfer of electrons from PSII to PSI and to pump protons across the thylakoid membrane via a plastoquinone-mediated Q cycle, analogous to that found in mitochondrial e transport (Chapter 21). Cytochrome f (f from the Latin folium, meaning “foliage”) is a c-type cytochrome, with an -absorbance band at 553 nm and a reduction potential of 0.365 V. Cytochrome b6 apparently does not lie directly on the pathway of electron transfer from PSII to PSI. This cytochrome, whose -absorbance band lies at 559 nm and whose standard reduction potential is 0.06 V, is thought to participate in an alternative c y c l i c e transfer pathway. Under certain conditions, electrons derived from P700* are not passed on to NADP but instead cycle down an alternative path via ferredoxins in the PSI complex to cytochrome b6, plastoquinone, and ultimately back to P700 . This cyclic flow yields no O2 evolution or NADP reduction but can lead to ATP synthesis via so-called cyclic photophosphorylation, discussed later.

Electron Transfer from the Cytochrome b6/Cytochrome f Complex to PSI

Plastocyanin (“PC” in Figure 22.12) is an electron carrier capable of diffusion along the inside of the thylakoid and migration in and out of the membrane, aptly suited to its role in shuttling electrons between the cytochrome b6/cytochrome f complex and PSI. Plastocyanin is a low-molecular-weight (10.4 kD) protein containing a single copper atom. PC functions as a single-electron car-

rier ( 0.32 V) as its copper atom undergoes alternate oxidation–reduc-

2

tion between the cuprous (Cu ) and cupric (Cu ) states. PSI is a light-driven plastocyanin:ferredoxin oxidoreductase. When P700, the specialized chlorophyll a dimer of PSI, is excited by light and oxidized by transferring its e to an adjacent chlorophyll a molecule that serves as its immediate e acceptor, P700 is formed. (The standard reduction potential for the P700 /P700 couple lies near 0.45 V.) P700 readily gains an electron from plastocyanin.

The immediate electron acceptor for P700* is a special molecule of chlorophyll. This unique Chl a (A0) rapidly passes the electron to a specialized quinone (A1), which in turn passes the e to the first in a series of membranebound ferredoxins (Fd, Chapter 21). This Fd series ends with a soluble form of ferredoxin, Fds, which serves as the immediate electron donor to the flavoprotein (Fp) that catalyzes NADP reduction, namely, ferredoxin:NADP reductase.

The Initial Events in Photosynthesis Are Very

Rapid Electron-Transfer Reactions

Electron transfer from P680 to Q and from P700 to Fd occurs on a pico- second-to-microsecond time scale. The necessity for such rapid reaction becomes obvious when one realizes that light-induced Chl excitation followed by electron transfer leads to separation of opposite charges in close proximity, as in P700 :A0 . Accordingly, subsequent electron transfer reactions occur rapidly in order to shuttle the electron away quickly, before the wasteful back reaction of charge recombination (and dissipation of excitation energy), as in return to P700:A0, can happen.

What molecular architecture couples the absorption of light energy to rapid electron-transfer events, in turn coupling these e transfers to proton translocations so that ATP synthesis is possible? Part of the answer to this question lies in the membrane-associated nature of the photosystems. Membrane proteins have been difficult to study due to their insolubility in the usual aqueous solvents employed in protein biochemistry. A major breakthrough occurred in 1984 when Johann Deisenhofer, Hartmut Michel, and Robert Huber reported the first X-ray crystallographic analysis of a membrane protein. To the great benefit of photosynthesis research, this protein was the reaction center from the photosynthetic purple bacterium Rhodopseudomonas viridis. This research earned these three scientists the 1984 Nobel Prize in chemistry.

Structure of the R. viridis Photosynthetic Reaction Center

Rhodopseudomonas viridis is a photosynthetic prokaryote with a single type of photosystem. The reaction center (145 kD) of the R. viridis photosystem is localized in the plasma membrane of these photosynthetic bacteria and is composed of four different polypeptides, designated L (273 amino acid residues), M (323 residues), H (258 residues), and cytochrome (333 amino acid residues). L and M each consist of five membrane-spanning -helical segments; H has one such helix, the majority of the protein forming a globular domain in the cytoplasm (Figure 22.16). The cytochrome subunit contains four heme groups; the N- terminal amino acid of this protein is cysteine. This cytochrome is anchored to the periplasmic face of the membrane via the hydrophobic chains of two fatty acid groups that are esterified to a glyceryl moiety joined via a thioether bond to the Cys (Figure 22.16). L and M each bear two bacteriochlorophyll molecules (the bacterial version of Chl) and one bacteriopheophytin. L also has a bound quinone molecule, QA. Together, L and M coordinate an Fe atom. The photochemically active species of the R. viridis reaction center, P870, is composed of two bacteriochlorophylls, one contributed by L and the other by M.

Photosynthetic Electron Transfer in the R. viridis Reaction Center

The prosthetic groups of the R. viridis reaction center (P870, BChl, BPheo, and the bound quinones) are fixed in a spatial relationship to one another that favors photosynthetic e transfer (Figure 22.16). Photoexcitation of P870 (creation of P870*) leads to e loss (P870 ) via electron transfer to the nearby bacteriochlorophyll (BChl).The e is then transferred via the L bacteriopheophytin (BPheo) to Q A, which is also an L prosthetic group. The corresponding site on M is occupied by a loosely bound quinone, Q B, and electron transfer from Q A to Q B takes place. An interesting aspect of the system is that no elec-

Cytochrome with

4 heme groups

hν

H

Note: The cytochrome subunit is membraneassociated via a diacylglycerol moiety on its N-terminal Cys residue:

O

+ H H3N C C

CH2

FIGURE 22.16 ● Model of the structure and activity of the R. viridis reaction center. Four polypeptides (designated cytochrome, M, L, and H) make up the reaction center, an integral membrane complex. The cytochrome maintains its association with the membrane via a diacylglyceryl group linked to its N-terminal Cys residue by a thioether bond. M and L both consist of five membrane-spanning -helices; H has a single membranespanning -helix. The prosthetic groups are spatially situated so that rapid e transfer from P870* to Q B is facilitated. Photoexcitation of P870 leads in less than 1 picosecond (psec) to reduction of the L-branch BChl only. P870 is re-reduced via an e provided through the heme groups of the cytochrome.

C O C O

Membrane

anchor

Bacterial

F1F0 ATP synthase

726 Chapter 22 ● Photosynthesis

● The molecular architecture of PSI. PsaA and PsaB constitute the reaction center dimer, an integral membrane complex; P700 is located at the lumenal side of this dimer. PsaC, which bears Fe-S centers FA and FB, and PsaD, the interaction site for ferredoxin, are on the stromal side of the thylakoid membrane. PsaF, which provides the plastocyanin interaction site, is on the lumenal side.

(Adapted from Golbeck, J. H., 1992. Annual Review of Plant Physiology and Plant Molecular Biology 43:293–324.)

The Molecular Architecture of PSI

The structure of PSI from the cyanobacterium Synechococcus elongatus has been solved by X-ray crystallography, and this structure shows strong similarities to the R. viridis reaction center and our emerging view of the eukaryotic PSII, both discussed earlier. Because of direct correlations with information about eukaryotic PSI, this cyanobacterial PSI provides a general model for all P700dependent photosystems (Figure 22.20). Although PSI consists of 11 different protein subunits, all the electron-transferring prosthetic groups essential to PSI function are localized to just three polypeptides. Two of these, PsaA and PsaB (83 kD each), compose the reaction center heterodimer, a structural pattern that now seems universal in photosynthetic reaction centers. PsaA and PsaB each have 11 transmembrane -helices. PSI has approximately 100 chlorophyll molecules, including the two composing P700 and two positioned some 16 Å from P700, one of which functions as A0, the immediate e acceptor for P700 (Figure 22.20). Quinones are found in association with PSI, including one that functions as A1, an intermediate e carrier. The Fe-S center designated Fx bridges PsaA and PsaB. The third protein, PsaC (9 kD), bears two additional Fe-S clusters designated FA and FB; PsaC (along with two other proteins designated PsaD and PsaE) lies on the stromal face of the reaction center complex. PsaD is the site of ferredoxin binding in eukaryotic PSI systems. PsaF, with three transmembrane -helices, provides the interaction site for plastocyanin (on the lumenal side of the membrane).

The overall structure of S. elongatus PSI thus consists of a core reaction center surrounded by and connected to a large Chl-based antenna system. Three equivalents of such PSI complexes occur together to form a trimeric structure. Photochemistry begins with exciton absorption at P700, almost instantaneous electron transfer and charge separation (P700 :A0 ), followed by transfer of the electron from A0 to A1 and on to Fx and then FA/FB, where it is used to reduce a ferredoxin molecule at the “stromal” side of the membrane. The positive charge at P700 and the e at FA/FB represent a charge separation across the membrane, an energized condition created by light.

22.6 ● The Quantum Yield of Photosynthesis

The quantum yield of photosynthesis, the amount of product formed per equivalent of light input, has traditionally been expressed as the ratio of CO2 fixed or O2 evolved per quantum absorbed. At each reaction center, one photon or quantum yields one electron. Interestingly, an overall stoichiometry of one H translocated into the thylakoid vesicle for each photon has also been observed. Two photons per center would allow a pair of electrons to flow from H2O to NADP (Figure 22.12), resulting in the formation of 1 NADPH and 12 O2. If one ATP were formed for every 3 H translocated during photosynthetic electron transport, 113 ATP would be synthesized. More appropriately, 4 h per center (8 quanta total) would drive the evolution of 1 O2, the reduction of 2 NADP , and the phosphorylation of 223 ATP.

The energy of a photon depends on its wavelength, according to the equation E h hc/ , where E is energy, c is the speed of light, and is its wavelength. Expressed in molar terms, an Einstein is the amount of energy in Avogadro’s number of photons: E Nhc/ . Light of 700-nm wavelength is the longest-wavelength and the lowest-energy light acting in the eukaryotic photosystems discussed here. An Einstein of 700-nm light is equivalent in energy to approximately 170 kJ. Eight Einsteins of this light, 1360 kJ, theoretically generate 2 moles of NADPH, 223 moles of ATP, and 1 mole of O2.

Experimental proof that the mechanism of photophosphorylation is chemiosmotic was provided in an elegant experiment by Andre Jagendorf and Ernest Uribe in 1966 (see figure). Jagendorf and Uribe reasoned that if photophosphorylation were indeed driven by an electrochemical gradient established by photosynthetic electron transfer reactions, they might artificially generate such a gradient by first incubating chloroplasts in an acid bath in the dark and then quickly raising the pH of the external medium. The resulting inequality in hydrogen ion electrochemical activity across the membrane should mimic the conditions normally found upon illumination of chloroplasts and should provide the energized condition necessary to drive ATP formation. To test this interpretation, Jagendorf and Uribe bathed isolated chloroplasts in a weakly acidic (pH 4) medium for 60 seconds, allowing the pH inside the chloroplasts to equilibrate with the external medium. The pH of the solution was then quickly raised to slightly

alkaline pH (pH 8), artificially creating a pH gradient across the thylakoid membranes. When ADP and Pi were added, ATP synthesis was observed as the pH gradient collapsed. This classic experiment was the first real proof of Mitchell’s chemiosmotic hypothesis and directed the scientific community to a greater acceptance of Mitchell’s interpretations. Mitchell’s chemiosmotic hypothesis for ATP synthesis now occupies the position of dogma as the weight of evidence has accumulated in its favor. Photophosphorylation then can be concisely summarized by noting that thylakoid vesicles accumulate H upon illumination and that the electrochemical gradient thus created represents an energized state that can be tapped to drive ATP synthesis. Collapse of the gradient—that is, equilibration of the ion concentration difference across the membrane—is the energy-transducing mechanism: the chemical potential of a concentration difference is transduced into synthesis of ATP.