Photosynthesis and Carbon Dioxide Fixation Muriel B. Bishop and Carl B. Bishop Clemson University, Clemson. SC 29634
Photosynthesis is the system of reactions by which higher plants, some algae, and bacteria capture solar energy, convert it to chemical enerev. and use this enerw in the reduction of carbon dioxide% sugars. The absorption of light enerav oiaments. the transfer of the ener-.hv" vhotosvnthetic . molec~l& a n d the stabilization of the gy among energy by charge separation are often referred to as the "light reactions"of photosynthesis. The products of the light reactions. stored as reducina vower and as chemical enerm in formof the compounds~~cotinamide adenine dinucleotide phos~hate (NADPH) and adenosine triphosphate (ATP), . . respectively, are used in the reduction o r fixation of the carbon source, carbon dioxide. The biochemical processes involved in carbon dioxide fixation are not directly light dependent and are called "dark reactions". The primary goal of this resource paper is to encourage the use of the concepts of photosynthesis to illustrate topics such as light energy, electronic transitions and spectra, electrochemistry, photochemistry, and metabolism, as they are taught in basic courses in chemistry, biology, and physics. This paper will emphasize carbon dioxide fixation and its dependence upon the light reactions. A paper emphasizing the energy transformations in the light reactions of photosynthesis appeared recently ( 1 ) in this Jownal. A summary ofthe lightreactions will appear in this paper as an introduction to carbon dioxide fixation. Photosynthesis occurs in the cells of green plants in a special organelle called the chloroplast. The chloroplast is an ellipsoid-shaped structure enclosed in a double layer of membranes. The outer membrane is permeable to most low molecular weight molecules. The inner membrane is selectivelypermeable to a few molecules, most of which are transported across the membrane by active transport proteins. The liquid that surrounds the inner membrane is called the stroma. The enzymes that catalyze the dark reactions are located in the stroma. Electron micrographs show structures, called thylakoids, that are formed from the internal membrane. Thylakoids appear as stacked, disc-shaped hodies, 5-10 pm in diameter, called grana. The light-absorbing pigment, chlorophyll, is bound to thelipid-protein complexes in the thylakoid and is responsible for harvesting and stabilizing solar energy. The proteins, which are active in electron transport, are also found in the inner membrane. Photosynthetic Plgments The primary light-absorbing pigment in all photosynthesizing plants is chlorophyll a (chl a). Chlorophyll is a magnesium-containing porphyrin structure similar to the iron-containing porphyrin structure of heme in the hemoglobin of blood. Chlorophyll contains a long, nonpolar hydrocarbon chain called a phytyl group. The phytyl group associates with nonpolar lipids in the cell membranes. Chlorophyll found in photosynthetic bacteria is slightly different in structure and is called bacteriochlorophyll a (Bchl a). Accessory molecules, such as chlorophyll b (chl b), carotenoids, and phycobilins, enhance the ability of the plant to harvest solar energy Over a spectrum. Structures of a, h, and Bchl a are shown in Figure 1. The chemical characteristics of the pigments determine the specific wavelength and the specific amount of energy 302
Journal of Chemical Education
absorbed. Chlorophyll a and h appear green because they absorb red light and transmit green light. The absorption of radiant energy causes the excitation of an electron in the radiated pigment, raising the electron from its ground state energy level to a higher energy level. There are several options by which the excited molecule may handle the excitation energy. First, the energy may he reemitted as aquantum of light energy (fluorescence). Second, the excitation energy may be transferred within a group of identical molecules or between groups of slightly different molecules. Third, the absorbed quantum of energy may be broken down into smaller auanta and reemitted as heat. Most of the solar energy absorbed by the photosensitive pigments is transferred among other chlorophyll molecules, but a small fraction of excitation energy is reemitted as fluorescence. The dependence of the excitation and emission spectra upon the chemical nature of absorbing pigment and the emitting pigment, and the mechanism of energy transfer can be used tocharacterize the absorbing pigment and to monitor the flow of excitation energy through a system of molecules. The spectral properties of the small amount of fluorescence emitted during energy transfer are used as a sensitive probe for studying the kinetics of the excited state. The term photosystem is wed to identify two distinyuishable amaratuses ralled I'hotosvsrcm 1 (1'51) and Photosyse as different &uctural entities within the tern 1f ~ h e s exist
X
GU
U 3 C-
\ /
9R Chlorophyll
,C.H
x
HZ[, cH, ; CHO
;
(rhl a ) (Chl
b)
/CH3 /
HZC,
H,C/
Cvi ,H C '~11,
Figure 1. The structure of chlorophyll. Chl a has a methyl group at position 3. whereas chl b hasan aldehyde group in that position. Bchi a differs from chl a in the degree of unsaturation of me 11 ring. Bacteriapheophytln( ~ p h e ahas ) no magnesium.
W
w H
6 NAOPH
Figure 2. The Calvin palhway forcarban dioxide fixation. The small dark circles represent carbon atoms, and P represents the phosphategroup. The fallowing abbreviations are used: RuBP = ribulosel.5-bisphosphate; PGA = 3-phasphoglyceric acid; DPGA = 1.3diphosphoglyceric acid; GAP = glyceraldehyde3-phosphate; DHAP = dihydroxyacetane phosphate; SBP = sedoheptulose-1.7-bisphosphate; S7P = sedoheptulose7-phosphate: FBP = fructose-1.6bisphasphste; F6P = fructose-6phosphate: Xu5P = xylulose5-phosphate; R5P = ribosed-phosphate; Ru5P = ribulose-5-phosphate.
chloroplast membrane and are both involved in harvesting solar energy and bringing about charge separation. Two functionally and spectrally different types of pigment-protein complexes make up the structural unit of a photosystem. The hulk of the chlorophyll molecules and accessory pigments of a photosystem is located in light-harvesting antenna called photoreceptor centers. The function of this first type of pigment complex is to absorb photons of light and transfer the resulting electronic excitation to the second type of pigment complex, called the photochemical reaction center, where energy is stabilized by oxidation-reduction actions. The light-gathering pigments, the chlorophyll molecule at the reaction center, and the components of the electron transport system must he embedded close together in the lipid-protein complex of the thylakoid membrane in order for the maximum excitation energy transfer to occur in the short period of time of the excited state of the emitting molecule. This close proximity of the molecules in the photo-
system assures that only a very small fraction of light energy is lost as fluorescence or heat. Only photosynthetic organisms can utilize solar energy to extract electrons from a weak donor such as water and use the hydrogen and electrons to reduce carbon from the level in carbon dioxide to that in sugars.
The fixation of carbon dioxide requires that the two systems of light reactions operate in a cooperative manner to give a continuous source of ATP and NADPH.
The net result of photosynthesis in plants and algae is the formation of reduced carbon compounds and molecular oxygen. Volume 64
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April 1987
303
Calvln Cvcle Although it has been known for over a century and a half that plants use solar energy in the fixation of carbon dioxide, it was not until the discovery of the long-lived carbon-14 i s o t o ~ ein 1940 that the Dathway of reductive carboxylation was &cidated. Melvin Calvin and others (2) found that, when plants supplied with '4C02 were irradiated with light, the threecarhon acid, 3-phosphoglycerate (PGA),was the first detectable product containing radioactive I4C. By using chromatography and autoradiography they were able to isolate and identifv the labeled intermediates and delineate the sequencd in which these i n t r r m d ~ a t e were s synthesized. This nnthwav of rarhon dinxide fixarion is known either as the kalvin Eycle or as the reductive pentose phosphate (RPP) pathway or the CQcycle. The biochemical processes of the Calvin cycle shown in Figure 2 are common to green plants and a variety of photosynthetic bacteria. The cycle may be conveniently divided into four parts. (I) Initially a carboxylation reaction fixes C02 into an acceptor molecule, rihulose-1,5-hisphosphate (RuBP). The enzyme that catalyzes this reaction, rihulose bisphosphate carboxylase, makes up 20-30% of all plant protein, thus making it the most abundant protein on earth. The carboxylated RuBP is hydrolytically split (Fig. 2, step 1) into two molecules of the three-carbon acid, 3-phosphoglvcerate . ,~~ ~.(PGA). In the second (11) Dart of the ~ a t h w a vthe mrhondiuxidr; which has been fixch at the oxidation le\;el of an arid tPGAr. is reduced (stev?I to the level of an aldehyde, g~~ceral'dehydk-3-phosphate ?GAP). This occurs through the phosphorylation of PGA with adenosine triphosphate (ATP) to give 1,3-diphosphoglycerate (DPGA). DPGA is reduced to glyceraldehyde-3-phosphate (GAP) by nicotinamide adenine dinucleotide phosphate (NADPH) (step 3). The net change for the production of six molecules of GAP is ~
~~
~
~
-
3RuBP + 3C0,
+ 6NADPH + 6H' + GATP 6GAP + 6NADP + 6ADP + 6Pj + 3H,O (3) = adenosine dinhos~hate . . and P: = inorganic
where ADP phosphate, HPOs02-. All three of the above C* intermediates are also found in the ylycolytic cycle where suyars are oxidized ro acids. However, in rhe third Dart (Ill) ofthe Calvin rvcle the flow of the Cg carhon compo;nds in carhon fixation istoward reduction. BY isomerizations (Fia. 2, steps 4 and 11). condensations (sieps 5 and I ) , and hydrolytic (steps 8 and 9) and rearrangement reactions (steps 6 and 101, five of the six molecules of GAP are converted to three molecules of pentose sugars and ultimately to three molecules of ribulose-5-phnsphate (RuSP).
Finally (IV), phosphorylation of Ru5P with ATP (step 12) regenerates the original acceptor molecule, RuBP, which begins the cycle again. Six molecules of C O ~ m u she t fixed for the net synthesis of the molecule of glucose as shown below. 6C0,
-
+ IZNADPH, + IBATP 1glucose + 12NADP + 18ADP + 18Pi + 6Hz0
(6)
The Calvin cycle as summarized in eq 6 is used universally in all photosynthetic organisms for the reduction and processing of fixed carbon.
The Hatch-Slack Pathway A large number of plants and bacteria fix carbon solely by the Calvin cycle and produce the same initial CQacid, PGA. However, Kortschak (3)discovered that the first products of I4CO2fixation in sugar cane plants are the C4 acids, oxaloacetate (OAA), malate (Mal), and aspartate (Asp), rather than 304
Journal of Chemical Education
Figure 3. The Hatch-Slack or C, pathway. The abbreviations used are as follows: OAA = oxaloacetate: Msl = malate: Asp = aspartate; Pyr = pyruvate: PEP = phosphoenol pyruvate.
PGA. The steps in carhon dioxide fixation by way of the Cp acids (Fig. 3) were elucidated by Hatch and Slack (4). The enzymes of the C4 or Hatch-Slack pathway are found in a t least 18 plant families. Generally, these families of plants also contain the Cg enzymes. C4 plants, such as corn, sugar cane, and some weeds, are able to photosynthesize a t high rates in hot, arid climates under conditions of bright light, low humidity, and warm temperatures. C4 plants must take up COz at a rate to maintain photosynthesis while simultaneously conserving water. The openings on the leaves (stoma) through which carhon dioxide enters remain partially closed during the day in order to reduce the water loss. The hiochemical mechanism for net carhon dioxide fixation in C4plantsinvolves differences in the leaf anatomy and in the biochemical reactions. The separate compartmentalization of the enzymes of the C4 cycle from those of the Ca pathway insures that CO2 fixation by the C4 pathway occurs prior to the Cg pathway. Figure 3 shows the steps of the C4 ~ a t h w a vand the relations hi^ of this pathwav to the Calvin or C3 cycle. The initial fixation of carhon dioxide bv the Ca pathway occurs in the chloroplasts of the mesophyll cells, which have reads access to atmospheric carbon dioxide since they occupy the most external location in the leaf. The first e n h e in the C4 pathway, phosphoenol pyruvate carhoxylase (PEPase), catalyzes the carboxylation of a three-carbon acceptor, phosphoenol pyruvate (PEP), to give the four-carbon compound, oxaloacetate (OAA). PEP3-
+ HC0;-
-
OAAZ- + P-:
(7)
Ox~loacctateis rapidly redured hy NADPH ro either of the four-carhon acids, malate (Val) or aspartaw (Asp). l'roduction of aspartate also requires the uptake of ammonia. For simplicity only the decarhoxylation of Ma1 will he followed. Both Mal and Asp diffuse out of the mesophyll cells and into the hundle sheath cells. There the same carbon that entered the cycle initially as COz is lost as COz from Mal. The carbon dioxide is then used in the Calvin cycle reactions to synthesize PGA and other materials. Carhon metabolism by the Cg pathway takes place in the chloroplasts of the internal bundle sheath cells found in close proximity to the vascular system which transports the suaars to other parts of the plant. ?hemain function of the c4pathway appears to be to take UP carhon dioxide from the atmosphere through the stomata of the leaves and to pump the rarhon dioxide hy way of fourrarhon compounds into the inner rells where it is passed to the Cs cyclewith little danger of being returned to the atmosphere. This mechanism insures a high Con concentration a t the site of the RuBP carhoxylase in the bundle sheath cells in spite of the unfavorable conditions of carbon dioxide uptake a t partially closed stomata.
The leavesofapeciea of plants with C4metabolism areahle to assemhle carhnn dioxide a t ahnut twice the rate of C1 plantn. As a result C, plants have two to three times the seasonal growth ratesof thnse plants that use only the Calvin cvrle mechanism. A second modification of the method of carhnn dioxide fixation, called crasaulacean acid metahnlism (CAM). has h e n found in various families of hieher olantn. The CAM plant seems to he adapted for m a x k a i water conservation. thus nermittine.. olant erowih and ~ h o l n s v n . thesis in extremely arid environments. Gas exchange is a ncrturnal artivitv in these nlants. The stomata are cloned in the daytime nnicarhnn dioxide enters the leaves at night and is stored as PEP. The pineapple in one of the few importnnt CAM plants. CAM plants are a striking example of regulation of photosynthetic productivity a t a level that permits growth and survival in totally unsuitable ennronments. Pholmrpk*
All plantn are metabnlically active even when sunlight is not availahle. In the dark they ohtain energy by oxidizing ~hotnsvntheticproducts hark to carbon dioxide. This nro&s is GRIM "dark respiration". ~ uC3t rJlan~?also carry out photorespiration, a light-dependent release of CO? and consumption of 0 2 . which decreases the efficiency of overall phntoaynthetic carhnn dioxide fixation. Photorespiration involves competition hetween carhon dioxide and oxygen for rihulme-1.5-hisphosphate carhoxylase, the carbon dioxide fixine.. enzvme of the Calvin cvrle. At hieh . " carhnn dioxide and low oxygen levels the enzyme acts as a carhnxylase and catalyzes the incorporation of carbon dioxide to give HuRI'. At low carhnn dioxide and high oxygen levels, however, the pnme enzyme displays oxypenase activity that leads to the oxygen consumption and cnrhnn dioxide evolution (photorespiration). Appreciable light-enhanced utilization of oxygen isabsent in C , plants. Photosynthesis in C, plants,unlike in Caplants. is not inhihitpd hy increased oxygen level and high temperatures. An explanation for the apparent lack ofphotorespiration and oxygen inhihition is that any carhnn dioxide evolved by photorespiration is virtually assured of heing refixed hy the C4 pathway, therehy maintaining a high enough C02/O? ratio in the bundle sheath cells that would allow the carbon dioxide to mmnete with the oxveen for the RuHP carhnxylase. Similarly the increased car& dioxide level would decrease the RuHP oxveenane activitv. Photorespiration may actually de'a protective t k h a n i s m aminst photmxidative damnae of the rhloro~lantundercondjtions of low carhnn dioxide supply.
Utilizatinn of solar energy by plants will need to he improved in order to n u ~ n l vthe nutritional needs of the world's increasing popuiitim. Measurements shnw thnt 2.5 X 10'" kcal of solar energy reach the earth's surface each year. Approximately 1.5 X 10:" kcal of this enerp?. is actually ahsorhed hy plants. Fdtimations on the perfect conversion of this energy into chemical energy as glucose would shnw six million tons of glucose formed per second. The earth's plant life falls far short ofthe maximum yield. An estimateof only 0.17~of solar radiation converted to chemical energy stored in plant matter ia more reaaonahle. The ahility of C4 and CAM plants to fix carhon dioxide ravidlv . . at hieh temneratures and to conserve water has heen of interest to plant scientists over the yearn. Attempts to improve individual suhromnonents of cron ~hotnsvnthetic attiihutes into ~ ~ ' ~ i of n closely n ~ s systems hy hreeding related speries have heen unsurressful. Housever, recent research has indicated thnt photorespiration can be reduced hy altering the kinetic properties of RuDP carhnxvlase/nxvgenase and through use , f carbon dioxide concentrating mechanisms. The increasing interest in the utilization of arid land for prnwing agronomically useful plants will continue to stimulate research into photosynthesis in plants that are ahle to mow under unfavorahle conditions. Our increasin'g knowledge of the difference h t w e e n C3 and C4 phntnayntherir could also lead t o t he development of new herhicidea. Current aprirultural practices rely heavily on the use of herhirides thnt kill C, weeds, such as nutgrass and Johnson grass, and leave C : , c r o p unharmed. The siteof action of mmt of these herhirides is unknown. The products of fermentation of cnrhohydrates and of wood are major renewahle, nonfwsil energy sources in some countries and are hyprnrlurts of photosynthesis just as is food. If plants grown as renewahle energy sources are to use land that rnuld he used to produce food, then the efficiency of phntnsynthetic enerp?r conversion must he increased in order that food production meet world needs. Other projects of research on photosynthesis include the use of genetically modified algae or artificial memhranes to manipulate photnsynthesis for the production of hydrogen. Certain research projects on increasing the yield of harverted solar energy have utilized models ofphotnsynthetic systems. A group in Japan has made semihinloeirnl nhotorells to generate dectririty directly from sunligh;. ~ h & photne cells contain tin oxide electrodes coated with chlorophyll and phmpholipids.
(.,
phococ*mwt*wkprorwrrmt Improving photmyntheais in plants and artificial chemical systems has hecome the incus of a major research effort.
Volume 84
Nvmkn 4
April 1987
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