EFB325 Cell Physiology

Photosynthesis

Photosynthesis is the process by which plants and algae capture light energy and use it to generate reduced carbon compounds (sugars and starch) which a chemotroph will use as food. Plants and algae are phototrophs. The synthesis of sugars occurs using the waste products of respiration, CO₂ and H₂O, to generate sugars and O₂.

The light reactions (photophosphorylation)

In overview:

• light energy is captured by pigment-protein complexes in the thylakoid membrane, this energy drives electron transport through membrane-bound proteins, the oxidation of water provides the source of electrons, which eventually are used to reduce NADP⁺ to NADPH
• the transport of electrons drives the transport of H⁺ into the lumen and "splitting" water releases H⁺ in the thylakoid lumen, generating a H⁺ electrochemical gradient, H⁺ flow down the gradient through the ATP synthase, driving the production of ATP in the stroma
• the ATP and NADPH produced by these reactions is then used by reactions in the carbon-fixation (C3 or Calvin) cycle to reduce CO₂ to sugars

Light for photosynthesis is captured by pigments, mostly by chlorophyll

• chlorophyll absorbs light of particular wavelengths better than other wavelengths (absorbs red and blue, reflects green)
• chlorophyll looks very much like heme, except it binds Mg, rather than Fe
• plants and algae have other pigments, some which absorb light for photosynthesis at other wavelengths

Electron transport is accomplished by multi-subunit, membrane-bound protein complexes

CLICK HERE TO SEE A FIGURE SHOWING PHOTOSYNTHETIC ELECTRON TRANSPORT
(Taiz and Zeiger, 2006, Plant Physiology 4 ed. - Fig. 7.22)

• two of these complexes bind reaction center chlorophylls that absorb light energy=photosystem II and photosystem I
• the third complex, the cytochrome b₆/f complex, transports electrons between the two photosystems
• electrons are "shuttled" between the three protein complexes by two mobile carriers, plastoquinone (PQ) and a small protein (plastocyanin)
• the full path of electron flow is from water to NADP⁺ , which is reduced to NADPH=non-cyclic (or linear) electron flow

Chlorophyll is bound to proteins in the chloroplast thylakoid membrane

• light energy is first captured in the antenna complexes, proteins with many chlorophyll molecules bound
• antenna chlorophylls pass energy to nearby pigment molecules through resonsance transfer
• the antenna complexes funnel light energy special chlorophyll molecules bound to the core of this large protein complex=reaction center
• once the reaction center receives light energy, it energizes the electron that was extracted from the oxidation of water
• the electrons are energized by two photons of light, one received in photosystem II and one in photosystem I

Photosystem II absorbs light energy, oxidizes water, and reduces plastoquinone

• the light energy harvested by the photosystem II reaction center generates an energized electron which is passed through a series of redox carriers bound to photosystem II, eventually reducing PQ (to PQH₂)
• once the photosystem II reaction center has lost an electron (it becomes Chl⁺), it becomes a very strong oxidizer, it oxidizes water in the lumen and thus is reduced (back to Chl), ready to absorb more light energy
• water oxidation =2H₂O -> 4H⁺ + O₂ + 4e^- (notice that this releases H⁺ in the lumen)

The cytochrome b₆/f complex acts to pump H⁺ across the thylakoid during electron transport

• the H⁺ used when PQ is reduced to PQH₂ come from the stroma
• reduced PQH₂ then passes electrons to the cytochrome b₆/f complex (PQH₂ is oxidized to PQ + 2H⁺)
• the cytochrome b₆/f complex releases H⁺ into the lumen (acting as a pump)
• the electrons pass through redox carriers in the protein complex, then are used to reduce a small protein, plastocyanin
• plastocyanin carries an electron using a bound copper atom and shuttles electrons to photosystem I

Photosystem I absorbs light energy, then reduces NADP⁺ (to NADPH) and oxidizes plastocyanin

• as with photosystem II, light energy is captured by the photosystem I and adds energy to an electron
• this energized electron is passed through a series of redox carriers on the protein, then is used to reduce NADP⁺ to NADPH (which removes a H⁺ from the stroma)
• the electron donated by the photosystem I reaction center is replaced by the electron carried by plastocyanin so that the reaction center can absorb light again

The process of light-driven electron transport and water oxidation generates a H⁺ gradient across the thylakoid membrane

• a higher concentration of H⁺ builds up inside the lumen, because H⁺ are released in water oxidation and H⁺ are pumped into the lumen by the cytochrome b₆/f complex
• this concentration difference represents potential energy

As in mitochondria, the flow of H⁺ through an ATP synthase protein drives ATP synthesis

• the CF_0-CF₁ ATP synthase protein in the thylakoid membrane is very similar to the ATP synthase in mitochondria
• the H⁺ flow from the lumen (inner-most compartment) to the stroma
• the flow of H⁺ drives synthesis of ATP in the stroma=photophosphorylation

Under some conditions, photosystem I can operate alone, with no reduction of NADP⁺=cyclic electron flow

• the energized electrons from the photosystem I reaction center are passed through carriers, then back to the cytochrome b₆/f complex
• while passing through the cytochrome b₆/f complex, PQ is reduced to PQH₂, then is oxidized back to PQ, pumping H⁺ from the stroma to the lumen
• the cytochrome b₆/f complex reduces plastocyanin, which will reduce the photosystem I reaction center, after it absorbs another photon of light
• this cycle pumps H⁺, which are used to generate ATP, but does not yield any NADPH

Concepts in common between photosynthetic and respiratory electron transport & ATP synthesis

• both involve sequential oxidation/reduction reactions between redox carriers on multi-subunit, membrane bound proteins
• some of those redox carriers are similar (hemes in cytochromes, Fe-S centers, Cu)
• both use 2 mobile electron shuttles to carry electrons between the protein complexes: a quinone and a small protein (cytochrome c in respiration; plastocyanin in photosynthesis)
• the energy released during the redox reactions of electron transport is used to pump H⁺ and generate a H⁺ gradient
• the H⁺ flow down the gradient through an ATP synthase (proteins are very similar in respiration and photosynthesis) which drives ATP synthesis

Note the differences:

• the direction of H⁺ transport is reversed within the organelle (H⁺ are pumped inward during photosynthesis and pumped outward during respiration)
• photosynthesis generates O₂, respiration consumes O₂
• photosynthesis requires 2 inputs of energy, respiration starts at the energy level of NADH and cascades down from there
• NADH is used in respiration, NADPH is the product of photosynthesis
• photosynthesis can operate cyclically, generating only ATP, not reducing currency (no NADPH)

The similarities provide strong evidence for a common evolutionary origin for photosynthetic and respiratory electron transport. There are present-day examples of more primitive forms of photosynthesis - such as green sulfur bacteria and other photosynthetic bacteria. These have more simple photosystems that perform cyclic electron transport, driven by light, to accomplish H⁺ pumping, which drives ATP synthesis by an ATP synthase.

The carbon-fixation (C3 or Calvin) cycle

The ATP and NADPH produced by photosynthetic electron transport (the light reactions) are now used to incorporate CO₂ into reduced carbon compounds in a pathway called the carbon-fixation cycle (discovered by Melvin Calvin in the 50's, he won the Nobel Prize in 1961).

The carbon-fixation cycle occurs in the stroma and can be broken down into three steps:

1) Carboxylation (catalyzed by Rubisco-see below)

2) Reduction (using ATP and NADPH from the light reactions)

3) Regeneration (needed to keep the cycle going)

Carboxylation combines CO₂ with a 5-carbon/2-phosphate sugar (ribulose-1,5-bisphosphate= RuBP) to produce 2 molecules of a 3-carbon/phosphate (3-phosphoglycerate)

• this reaction is catalyzed by the most abundant protein on Earth, Ribulose-1,5-bisphosphate Carboxylase/Oxygenase=Rubisco

The reduction step uses ATP and NADPH to reduce 3-phosphoglycerate to triose-phosphate

• this step is essentially the reverse of the oxidation step in glycolysis
• for every 3 CO₂ that are combined with 3 RuBP, 6 molecules of triose-phosphate are produced

For every 6 triose-phosphates produced (from three carboxylation steps), 1 is used for sucrose or starch synthesis and 5 are used to regenerate RuBP

• 5 triose-phosphates combine (in several steps) and 3 more ATPs are used to regenerate 3 RuBPs (5-carbons each) to keep the cycle going

The carbon-fixation cycle looks very much like the reverse of glycolysis (compare Fig 14-39, pg. 486 with Panel 13-1, pgs. 432-433)

• the intermediates are phosphorylated sugars and organic acids
• the reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate (using NADPH) is the reverse of the oxidation step in glycolysis that generates NADH
• the carbon-fixation cycle uses ATP, while glycolysis generates ATP by substrate-level phosphorylation

Rubisco can use O₂ instead of CO₂, resulting in an oxygenation reaction (instead of carboxylation)=photorespiration

• when RuBP combines with O₂, a 2-carbon/phosphate and a 3-carbon/phosphate are produced
• the 3-carbon/phosphate continues on in the carbon-fixation cycle
• the 2-carbon/phosphate is processed through the C2 cycle: it is exported to the peroxisome, then the mitochondria, where 2 2-carbon compounds combine to form a 3-carbon compound and release CO₂
• the 3-carbon compound moves back through the peroxisome to the chloroplast, where ATP is used to regenerate the carbon-fixation cycle intermediate
• for every 2 oxygenation reactions, one fixed CO₂ is lost (and some ATP is used up)
• the ratio of O₂ (21% of air) to CO₂ (0.035% of air) results in 1 oxygenation for every 3 carboxylations; greatly reducing the efficiency of photosynthesis

Triose-phosphate from the carbon-fixation cycle is used to make starch (in the stroma) or sucrose (in the cytosol)

• two triose-phosphates combine to form fructose-phosphate, which is converted to glucose-phosphate for starch or sucrose synthesis
• starch is a storage form of photosynthate, sucrose is the main transported sugar (it is also used for storage by some plants, like sugar beet and sugar cane)

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