Photosynthesis: Chloroplasts and Light

Photosynthesis: overall process involves the oxidation of water (removal of electrons
with release of O2) and the reduction of CO2 I. Historical summary of early photosynthesis research A. 1727: Stephen Hales: part of plant nourishment came from the atmosphere and
light B. 1771: Joseph Priestly: ident2 in the process of photosynthesis. Green
plants renewed "bad" air from breathing animals C. 1700's Jan Ingenhousz: light was necessary for purification of air D. 1782: Jean Senebier: gases 2) produced by plants and animals during the dark could stimulate production of "good air" (O2) in the light. E. 1804: N.T. deSaussure: noted the importance of water in photosynthesis. Found that a gain in plant weight could not be accounted for by O2 and CO2. F. 1864: Julius von Sachs: recorded organic matter synthesis by observing the growth of starch grains in illuminated chloroplasts. Starch is the most abundant photosynthetic product formed by chloroplasts. G. Early 1930's: C.B. van Niel: noted the similarity between green plants and bacteria photosynthetic processes. Found that O2 released by plants was derived from water not from CO2 nCO2 + 2nH2S + light => (CH2O)n + nH2O + 2nS H. Late 1930's Robin Hill and R. Scarisbrick: if suitable electron acceptors (Ferric, Fe3+, salts) where provided for accepting electrons removed from H2O,
isolated chloroplasts and chloroplast fragments could release O2 in the light. Photolysis: light driven spliting of water in the absence of CO2 fixation became known as the Hill Reaction. I. 1941: Samuel Ruben and Martin Kamen: using a heavy isotope of oxygen (18O2) in water, they found that the oxygen released was labeled, supporting Hill’s
hypothesis. J. 1975: Alan Stemler and Richard Radmer: final definite proof of oxygen coming
from water. nCO2 + 2nH2O + light (chloroplasts) => (CH2O)n + nH2O + 2nO K. 1951: Nicotinamide adenine dinucleotide phosphate (NADP+), a coenzyme containing vitamin B (niacin or nicotinamide) was found to be the electron acceptor in the Hill reaction. 1. Two essential functions of light in photosynthesis: a. drive electrons from H2O to reduce NADP+ to NADPH b. provide energy to form ATP from ADP and Pi L. 1954: Daniel Arnon (Univ. California, Berkeley) was able to synthesize ATP in isolated chloroplasts 1. Photosynthetic phosphorylation or Photophosphorylation 2. ATP and NADPH used in process of CO2 reduction and carbohydrate synthesis II. Chloroplasts: Structures and Photosynthetic Pigments A. Proplastids: derived from female gametophyt. Develop into chloroplasts when leaves and stems are formed by exposure to light. A mature leaf cell can contain several hundred chloroplasts. B. Chloroplast Structure: 1. Envelope: double-membrane system that surrounds and controls molecular traffic into and out of the chloroplast 2. Stroma - amorphous, gel-like, enzyme-rich material where CO2 is converted
to carbohydrate 3. Thylakoids - membrane structures that contain pigments (chlorophylls and carotenoids) where light energy is used to oxidize H2O and form the energy-rich ATP and NADPH 4. Grana (granum, singular) - thylakoid stacks a. Appressed Region - region where one granum thylakoid contacts another b. Nonappressed Region - region where a granum is exposed to the stroma 5. Stroma thylakoids - long thylakoids that connect grana to each other and extend through the stroma. 6. Lumen - cavity between the two membranes of each thylakoid granum. Filled with water and dissolved salts. C. Pigments: 1. Chlorophyll a and Chlorophyll b: tetrapyrrole ring is made of four pyrrole rings and causes the green color. The phytol tail (C20H39) is hydrophobic and common to both chorophylls. The phytol tail extends into the interior of the thylakoid membrane. a. chlorophyll a is blue-green b. chlorophyll b is yellow-green 2. Carotenoids: yellow to orange pigments. Also exist in the chloroplast envelope
(chlorophyll not found in the envelope). a. Carotenes: pure hydrocarbons. beta-carotene most abundant b. Xanthophylls: contain oxygen. Lutein most abundant 3. Chlorophylls and carotenoids are embedded within the thylakoid membranes and are attached by noncovalent bonds to protein molecules a. Chloroplast pigments represent about 1/2 of the lipid content of the thylakoid membranes. The other 1/2 are galactolipids with only small amounts of phospholipids. There are few sterols in the thylakoid membranes. b. Galactolipids and phospholipids make a bilayer. Fatty-acid portions of thylakoid
lipids are rich in linoleic acid (18:3) and linoleic acid (18:2). Unsaturated fatty acids cause thylakoid membranes to be unusually fluid. 4. DNA, RNA, Ribosomes, Enzymes: found in stroma a. Chloroplast DNA (cpDNA) 50 or more supercoiled double-stranded circles
per plastid. b. Plastid genes code for transfer-RNA molecules (about 30) and ribosomal -RNA
molecules (four). c. 85 other such genes code for proteins involved in transcription, translation,
and photosynthesis d. Most proteins in plastids are coded by nuclear genes. III. Some Principles of Light Absorption by Plants A. Light has both a wave and a particle nature B. Wave Nature: 1. Electromagnetic Spectrum 2. Visible Light: 390 to 760 nm [very narrow region of the electromagnetic spectrum] C. Particulate Nature: 1. Expressed in quanta or photons: a. Discrete packets of energy, each having a specific associated wavelength. b. Energy in each photon is inversely proportional to the wavelength. c. Einstein: one mole (6.02 X 1023) of photons D. Fundamental principle of light absorption: Stark Einstein Law 1. A molecule can absorb one photon at a time. 2. The photon causes excitation of only one electron. 3. Specific valence electrons in stable ground-state orbitals are excited. The
electrons are driven away from the ground state a distance corresponding to the energy exactly equal to the energy of the photon absorbed. 4. The pigment molecule in an excited state and its excitation energy are used
in photosynthesis. E. Chlorophylls and other pigments can remain in an excited state for only a
nanosecond. 1. Lost by heat release 2. Fluorescence: the production of light accompanying rapid decay of electrons in
the excited state. 3. Transferred between electron carriers F. Blue light on an energy basis is less efficient in photosynthesis than red light.
After excitation with a blue photon, the electron in a chlorophyll always decays
rapidly by heat release to a lower energy level. This is the same energy level an electron would obtain after absorption of a red photon but without the heat loss.
From this lower level, either additional heat loss, fluorescence, or photosynthesis
can occur. G. The energy in excited electrons of different pigments is transferred to a special
energy-collecting pigment: a reaction center. 1. Energy in one excited pigment can be transferred to another adjacent pigment 2. Energy migrates by exciton transfer through inductive resonance. H. Leaves absorb 90% of the violet, blue, orange, and red wavelengths. A graph of
absorption as a function of wavelength is called an absorption spectrum. I. Carotenoids: transfer their excitation energy to the same reaction centers as
chlorophylls. 1. Caroatenoids absorb blue and violet wavelengths in vitro. 2. Carotenoids function to as light-harvesting pigments in photosynthesis, and
protect chlorophylls against oxidative destruction by O2 under high irradiance
levels . J. Comparison of effects of different wavelengths on the rate of photosynthesis 1. Action Spectrum: used to identify pigments involved in photosynthesis. The
absorption spectrum of a pigment participating in photosynthesis should closely
match the action spectrum for photosynthesis. 2. Light absorption by chlorophylls and carotenoids results in a major peak in the
red-light region and a distinct lower peak in the blue-light region. 3. A note of interest: conifers show a lesser response to blue light than
angiosperms. This is because (1) the waxy needles of conifers reflect blue
light and (2) their higher amounts of carotenoids, which absorb blue light but do not transfer the energy to chlorophylls for photosynthesis. The Blue spruce
and the Colorado spruce species show little photosynthesis in blue and violet light. K. The ability of angiosperms to absorb and use green and yellow light to in
photosynthesis is high compared with the absorption spectra of purified
chlorophyll and carotenoids at these wavelengths. 1. The reason that the action spectra are higher than the absorption spectra for
yellow and green wavelengths is that only a small amount of light in these
wavelength are absorbed. Those wavelengths not absorbed are reflected from chloroplast to chloroplast until they are absprbed. L. In vivo carotenoid absorption shifts from the blue region of the spectrum into the
green region. Some photosynthesis occurs due to the absorption of carotenoids
in the green region at 500 nm. 1. Chlorophylls a and b show small shifts in vivo in the blue region. Chlorophyll a
exhibits several shifts in the red region caused by the association of chlorophyll
a with thylakoid proteins. 2. Reaction Centers: chlorophyll a molecules absorbing at 680nm and 700 nm in
special chemical environments. Abbreviated P680 and P700. IV. The Emerson Enhancement Effect: Cooperating Photosystems A. 1950’s Robert Emerson (University of Illinois) noted that red-light longer than
690 nm was unable to cause photosynthesis even though much of the red light
was absorbed by chlorophyll a in vivo. 1. However, if shorter wavelength light was provided at the same time as the longer
red light, the photosynthetic rate was greater than the sum of the two rates with
either color alone. This was called the Emerson Enhancement Effect. B. Turns out that the long red wavelengths help out the shorter wavelengths. 1. This lead to the discovery that two separate groups of pigments called
photosystems cooperate in photosynthesis 2. Long red wavelengths are absorbed only by photosystem I (PSI), whose reaction
center is P700. 3. Photosystem II (PSII) with reaction center P690 absorbs wavelengths shorter
than 690 nm. For maximum photosynthesis both systems must function together. 4. R. Hill and F. Bendall 1960: PSI and PSII use light energy to oxidize H2O and
cooperatively transfer the two available electrons in it to NADP+ thus forming NADPH V. Four Major Thylakoids Complexes A. Photosystem II (PSII) 1. This photosystem consists of a core complex of six integral polypeptides
connected non covalently to each other, and the reaction center P680. a. The polypeptides are coded by the chloroplast genome b. 33 kDa (D1) and 31 kDa (D2): directly bind P680 and quinones necessary for
the oxidation of water. c. Associated with PSII Core Complex and the membrane-lumen interface are
three peripheral polypeptides coded by nuclear genes. These polypeptides aid binding of Ca2+ and Cl- which are needed for photolysis of water. d. Also contains 40 molecules of chlorophyll a, several molecules of b-carotene,
membrane lipids (galactolipids), 4 manganese ions, one noncovalently bound iron, one or more Ca2+, several Cl-, two molecules of plastoquinone,
and two molecules of pheophytin. e. Plastoquinones carry two electrons from PSII to photosystem I, and transport
H+ from the stroma into the thylakoid lumen. f. Pheophytin (modified chlorophyll a molecule): two H+ atoms replace the central
Mg2+ of chlorophyll. g. PSII is present only in appressed regions of granna thylakoids. 2. P680 receives light energy by inductive resonance from about 250 chlorophyll a,
chlorophyll b, and xanthophylls. a. PSII Light Harvesting Complex (LHCII) b. Pigments are associated with integral proteins (10 chlorophylls, and 2 or
3 xanthophylls per protein molecule). c. Act as an antenna system to absorb light and pass the exciton energy to P680. d. Proteins in LHCII are encoded by nuclear DNA. Translated on cytoplasmic
ribosomes, and transported into the chloroplast thylakoids. e. PSII functions to use light energy in reduction of plastoquinone (PQ => PQH2)
using electrons from water. 3. Two water molecules (4 electrons) are needed to reduce each CO2, and two light
photons are required to oxidize each H2O. Summary of the function of PSII: 2H2O + 4 photons + 2PQ + 4H+ => O2 + 4H+ + 2PQH2 a. Show H+ on both sides of the equation because oxidation of water releases H+
in the thylakoid lumen, and reduction of PQ requires H+ taken from the opposite (stroma) side of the thylakoid. B. The cytochrome b6-cytochome f Complex (cytb6-f): 1. cytb6-f : four integral polypeptides a. Three contain iron: (1) reduction to Fe2+ (2) oxidation to Fe3+ b. First two polypeptides are cytb6 and cytochrome f ("frons" or leaf) (1) iron in a heme prosthetic group (2) genes in chloroplast c. Third Protein has two non-heme iron atoms (1) Iron is connected to two nonprotein sulfur atoms and two sulfur atoms of
cysteine residues in the protein (2) Iron-sulfur Protein (2Fe-2S protein) (3) gene for 2Fe-2S polypeptide is in the nucleus d. Fourth polypeptide: component IV, has no iron and no known function. (1) genes in chloroplast 2. cytb6-f complex exists in equal concentrations in grana and stroma thylakoids. a. Function is to pass electrons from PSII to PSI b. Oxidizing PQH2 and by reducing a small mobile, copper-containing
protein - Plastocyanin. c. Transport of H+ from the stroma into the thylakoid lumen d. Q cycle: Quinone. Increases the number of PQH2 molecules involved.
Doubles the amount of H+ transferred into they thylakoid lumen C. Photosystem I (PSI) 1. Absorbs light energy independently of PSII a. Core complex that receives electrons taken from H2O by the PSII core complex b. Eleven different polypeptides (1) six coded by nuclear genes, five coded by chloroplast genes (2) probably one of each polypeptide present per P700 reaction center. (3) Ia and Ib: two large polypeptides (a) coded in a single operon in the chloroplast genome (b) bound closely in thylakoids (heterodimer) (c) bind the reaction center P700 (d) with another polypeptide they also bind 50 - 100 chlorophyll a molecules,
b-carotene, and three electron carriers that help transport electrons toward NADP+ 2. A0, A1, X: Electron carriers associated with polypeptides Ia and Ib in the core
complex a. A0: chlorophyll a b. A1: phylloquinone (vitamin K1) c. X: iron-sulfur group 4Fe-4S attached to a 9 kDa polypeptide. Fe-S centers pick
up and transfer one electron at a time 3. PSI core complex receives light energy by inductive resonance from 100
molecules of chlorophyll a and b (ratio of 4:1). These chlorophyll are bound to
nuclear-endcoded proteins in the light-harvesting system along with the antennae pigment surrounding and core complex: LHCI. a. PSI is located in stroma thylakoids and nonappressed regions of grana that face
the stroma b. Oxidized plastocynanin, transfers electrons to Ferredoxin (Fe-S protein) (1) Ferredoxin: low-molecular weight protein (2) Peripheral protein attached to the stroma side of the thylakoids (3) 2Fe-2S cluster transfers one electron as one iron is reduced to F2+ Light + 4PC(Cu+) + 4Fd(Fe3+) => 4PC(Cu2+) + 4Fe(Fd2+) c. electrons from mobile ferredoxin are used in the final electron-transport step to
reduce NADP+ to NADPH + H+ d. Ferredoxin NADP+ reductase 4Fd(Fe2+) + 2NADP+ + 2H+ => 4Fd(Fe3+) + 2NADPH D. The ATP Synthase or Coupling Factor 1. ATP synthase: couples ATP formation to transport of electrons and H+ across
the thylakoid membrane (coupling factor) 2. Only in stroma thylakoids and the nonappressed regions of granna thylakoids. 3. Two major parts: a. Stalk: called CF0 extends from the lumen across the thylakoid membrane to the
stroma b. CF1: lies in the stroma. 4. Nine polypeptides exist in ATP synthase coded by chloroplast DNA and nuclear
DNA. 5. Similar to mitochondria extends form the lumen across the thylakoid membrane
to the stroma VI. Oxidation of H2O by Photosystem II: supply of electrons from the oxygen-evolving
complex A. Back reactions that would prevent oxidation of H2O are stopped by the stepwise
transfer of electrons completely across the thylakoid membrane starting from
H2O in the PSII core to those in the cyto b6-f core, then to those in PSI, and finally to ferredoxin and NADP+. B. Oxygen-Evolving Complex (OEC) 1. Light releases O2 from H2O in four distinct flash peaks 2. One O2 molecule requires oxidation of two H2O molecules and removal of four
electrons 3. A molecule accumulates a positive charge after each flash, until it accumulates
four positive charges and can receive four electrons back in a one-step oxidation
of two H2O molecules. 4. Water Oxidizing Clock 5. States represent various oxidation states of manganese, Mn2+ , Mn3+ , Mn4+ a. Four Mn atoms are associated with each PSII core system and all 4 are
essential for O2 release b. Bound to each other and to the D1 and D2 polypeptides on the thylakoid lumen
part of PSII where O2 is released VII. Electron Transport from H2O to NADP+ Across Thylakoids A. PSII and cyt b6-f complex are in the appressed thylakoid region. PSI and CF
complexes are in a stroma thylakoid or nonappressed region B. H2O is oxidized in the OEC, two electrons released for transport 1. Received one at a time by tyrosine amino acid in polypeptide D1. Tyrosine then
passes electrons to P680. 2. P680 accepts electrons only if it has lost an electron. Occurs when P680 has
been excited by light energy transferred to it from a light absorbing pigment in
LHCII a. Light oxidizes P680 b. P680+ acts as an electron attractant (oxidant) that pulls an electron from D1,
which in turn pulls an electron from Mn ion of the OEC c. P680 gives its electron to pheophytin (Pheo) 3. Pheo passes electron to plastoquinone (QA) a. QA attached to D2 b. QA passes electron to another plastoquinone (QB) (1) QA loosely attached to D1 4. Two electrons needed to reduce QA and QB a. Both electrons from H2O arrive one at a time b. Requires addition of two H+ B. Two electrons and two H+ required to reduce PQ is important for photosynthetic
phosphorylation. The H+ come from the stroma, when PQ is oxidized the H+ are
transferred into the thylakoid lumen. Result of PQ reduction by PSII and then
oxidation by cyt b6-f complex is the transport of two H+ from stroma to thylakoid
lumen. The H+ are transported back by ATP synthase in a process that drives
ATP formation. C. Steps involved in the electron transport: Some herbicides work by occupying the
QB-binding site on D1, this prevents biding of PQ and stops electron transport, thereby inhibiting photosynthesis. 1. For each pair of H2O molecules oxidized by OEC, four electrons are transported
through the quinones, so two QB molecules must be reduced, leave D1, and be replaced. 2. Each reduced QBs (PQH2) carries two electrons to the cyt b6-f complex, where
electron-transport process occurs. 3. cytb6-f complex accepts one electron at a time from each mobile PQH2 formed
in PSII complex. 4. Electrons pass, one at a time, to either Fe-S protein in the complex or to
cytob6. Fe3+ accepts electrons and is reduced to Fe2+. 5. Two H+ from each PQH2 are transfered into the thylakoid lumen 6. Each electron in the iron of cyt b6 and Fe-S protein is accepted by cyt f, which
reduces the iron to Fe2+ 7. cyt f donates an electron to Cu2+ in plastocyanin (PC), reducing it to Cu+. Cyt f
reduces four PC. 8. Each mobile PC carries an electron along the thylakoid lumen to PSI. 9. First molecule to accept is P700. P700 in PSI. Cannot accept an electron
unless it lost one. Loss of an electron by P700 to form P700+ occurs when light
energy is transferred from light-harvesting pigments in LHCI to P700. A photon
of light absorbed by PSI antenna pigment causes formation of P700+, which
takes an electron from reduced PC, forms oxidized PC and reduced P700.
Each electron from P700 moves to A0 (chlorophyll a) 10. A0 passes electron to A1 (phylloquinone vitamin K1) then to one iron in 4Fe-4S
protein in the PSI core complex. 11. Mobile ferredoxins (Fd) accept one electron and transfer it to NADP+ to form
NADPH in the stroma. D. Noncyclic Electron Transport: light-driven reactions by which electrons are
transferred across thylakoid membranes to form NADPH. E. Cyclic electron transport: electrons not donated to NADP+ by ferredoxin can be
transported to cyt b6-f complex. 1. Transfered directly to cyto b6 2. Move to plastoquinone, full reduction takes two electrons and two H+; both H+
come from stroma side of thylakoid. 3. As the electrons move to Fe-S protein, PQH2 releases two H+ into the thylakoid
lumen - contributs to the pH gradient 4. From the Fe-S protein electrons move via noncyclic pathway through cyt f and
plastocyanin, and back to P700. 5. Cyclic electron transport in PSI requires energy: one photon per each electron
transferred. F. One electron from H2O to NADP+ requires two photons because excitation of both
photosystems is essential. Explains Emerson Enhancement effect: cooperation
of two photosystems. G. For each molecule of CO2 fixed, one O2 is released and two H2O molecules are
used. 1. Number of photons of light is not specific. The number is important to calculate
photosynthetic efficiencies. 2. Model requires two photons for each electron transported 3. Minimum of eight photons required to oxidize two H2O molecules, release one
O2, and provide four electrons 4. Four electrons can reduce two NADP+, and two NADPH are essential to reduce
one CO2. 5. There is a discrepancy between the calculated eight photons needed and the
actual requirements of 9-12 photons. Three ATPs are needed to reduce one
CO2 to a simple carbohydrate. VIII. Photophosphorylation A. ATP synthase or coupling factor (CF0 + CF1) causes ATP formation in the stroma
and transport of H+ from the thylakoid lumen to the stroma 1. ATP formation requires H+ transport 2. H+ ions in the thylakoid lumen arise from oxidation of H2O and PQH2 3. H+ concentration in the lumen (pH 5) to become about 1,000X as great as that in
the stroma (pH 8) when photosynthesis is occurring. 4. Strong H+ concentration gradient toward the stroma, but thylakoids are
impermeable to H+ 5. Chemical-Potential Energy a. 1961, Peter Mitchell b. Chemiosmotic Theory: bears no clear relation to osmosis c. Uncouplers: remove the interdependence (coupling) of electron transport and
phosphorylation (NH3 and dinitrophenol). (1) Act as "ferryboats": move into thylakoid channels and pick up protons.
Carry protons back into the stroma, where the pH is higher and it reacts with
OH- to form H2O. (2) Gramicidin and carbonylcyanide p-trifluoromethoxyphenylhydrazone: block
photophosphorylation by preventing the exit of H+ through CF0. (3) Uncouplers destroy the pH gradient across the thylakoid membrane and
prevent ATP formation. 6. The number of H+ transported to form one ATP appears to be three. B. Oxidation of two H2O molecules will release four H+. Four more H+ arise from
two H2O molecules during noncyclic electron transport at the PQH2 oxidation step. 1. Eight photons are required to oxidize two H2O 2. Eight photons provide eight H+, not enough to form the more than three ATP
required to fix one CO2. 3. Noncyclic Photophosphorylation: process of ATP formation by these reactions
in which electrons from H2O are transported to NADP+, accompanied by H+
transport. 4. Cyclic Photophosphorylation: ATP is produced by ATP synthase in response to
the decreased pH in the thylakoid lumen C. If eight photons involve in both photosystems produce eight H+ by noncyclic
electron transport plus the four photons absorbed only by PSI,then twelve
photons total could give four ATP 1. Measurements in leaves show a minimum of nine (or 12) photons are required
for each CO2 used. 2. Both cyclic and noncylcic electron transport, give results consistent with the modle. 3. Minimum requirement of 12 photons CO2 + 2H2O + 12 photons => (CH2O) + O2 + H2O 4. ATP and NADPH do not appear in summary because their production is
balanced by their use in CO2 reduction. 5. If the Q cycle operates, the number of H+ moved from stroma to lumen during
electron transport is doubled. This increases the amount of photophosphorylation
(without changing the NADPH output) and could therefor lower the photon requirement to eight. IX. Distribution of Light Energy Between PSI and PSII A. PSII/PSI ratios vary from 0.43 to 4.1; the higher ratios obtained from plants in
deep shade. 1. Ratio of 1.0 seldom occurs B. Adaptation to light, both on a short-term and a long-term scale 1. Short term (30 seconds or less): photosystems adapt so that energy
redistribution occurs between PSII and PSI a. Movement of the LHCII pigments and proteins so that they associate with PSI
in stroma thylakoids b. LHCII pigments transfer more light energy to PSI and none to PSII c. Movement occurs because the proteins of LHCII become more negatively
charged by phosphorylation by a protein kinase that transfers phosphate form
ATP to proteins. d. Phosphate groups ionize (lose H+) to create the additional negative charges on
the proteins e. These negative charges force apart LHCII proteins with associated pigments.
They are then attracted toward a positively charged proteins of PSI in stroma
thylakoids. 2. Preferential absorption of light by PSII causes reduction of numerous PQ
molecules to PQH2 a. These PQH2 activate the protein kinase b. Light absorbed preferentially by PSI causes oxidation of PQH2 molecules,
stops phosphorylation and allows phosphate removal by a phosphatase that
hydrolyzes phosphate groups away form mobile LHCII proteins c. Loss of phosphate reduced their negative charge so that they move back to
appressed thylakoid regions and donate light energy to PSII.