Progress 10/01/06 to 06/30/08
Outputs
OUTPUTS: The information obtained during the 18 months of the project's duration have been disseminated mostly by email and phone discussions with colleagues in the field of photosynthesis. Although the project is officially terminated by the retirement of the PI as of July 2008, the results may continue to be disseminated in the future. PARTICIPANTS: Work was done in collaboration with Professor Emeritus Paul Castelfranco at UCD. Related work was done in collaboration with Galia Lazova at the Institute of Plant Physiology Acad. M Popov, Bulgarian Academy of Sciences, Acad. G. Bonchev str. Bl.21, Sofia 1113, Bulgaria TARGET AUDIENCES: The project can be classified as basic science with a world-wide audience of experts in the field of photosynthesis. Since the area of study, oxygen evolution, begins the process of energy capture by plants, it is relevant to the topic of alternative energy sources currently of national interest. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
In the recent past we published a model of photosynthetic oxygen evolution postulating a role for bicarbonate ions and their oxidized form, peroxydicarbonic acid, as chemical intermediates. The following summarizes our work of the past 18 months: 1) One requirement of our model is that photosystem II (PSII) must be able to catalytically hydrate CO2. That is, it must act as the enzyme carbonic anhydrase (CA). We discovered previously that there are two sources of CA activity in the PS II complex. Work on this project resulted in a discovery related to our assay for determining CA activity. To measure CA activity, our samples are stirred with a magnetic stirrer. Although not commonly recognized, a moving magnetic field can affect the chemical properties of water. This has been reported in a few journals and on the Internet, but the theoretical basis for the phenomenon has not yet been established. We found that our results were a function of the strength, distance, and exposure time of the magnetic field to our samples. The effect is not large, but it is clear from the literature that other researchers, although unaware, have observed this effect in the CA results that they reported. 2) After learning to control the magnetic field effect on our assay, we returned to the planned experiments studying PS II CA. While typical CA enzymes are inhibited by monovalent anions such as formate, the innermost PS II CA shows a complex relationship with this anion. At low pH and low formate concentrations, CA activity first increases, while higher concentrations inhibit as expected. To explain these results, we refer to information we gathered years ago. There is a pool of CO2/HCO3- bound to PS II. This pool, though tightly bound, can be removed at low pH and with the addition of formate. We therefore propose that the innermost PS II CA acts on this pool of tightly bound CO2/HCO3-. However, in vivo, the product of catalysis, HCO3-, is not released into the medium but is retained to be used in oxygen evolution. If this is the case, little CA activity will be measured because the assay requires continuous release of HCO3- into the medium. At low pH, however, addition of formate will initially cause the release of HCO3- into the medium thus resulting in an apparent increase in CA activity. High concentrations of formate, on the other hand, will cause the active site to be permanently occupied by formate. This will prevent the binding of substrate CO2 and thus inhibit CA activity. The presence of CA activity acting on a tightly bound pool of HCO3- on PS II provides additional support for our model of oxygen evolution. 3) We extended our studies of high-affinity bicarbonate binding to PSII. We used radiolabeled bicarbonate to determine that there is not one pool of tightly bound HCO3- on PSII, but two. This finding also supports our model of oxygen evolution. Our proposed intermediate, peroxydicarbonic acid, must be formed from two bicarbonate ions that continually recycle on the membrane. We propose that these tightly bound bicarbonate anions are at the active sites of the two carbonic anhydrases on PSII, exactly as predicted by our model.
Publications
- Lazova G N, Stemler A J, (2008) A 160 kDa protein with carbonic anhydrase activity is complexed with rubisco on the outer surface of thylakoids. Cell Biology International 32: 646-653
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Progress 01/01/07 to 12/31/07
Outputs
While we have worked continuously on the mechanism of photosynthetic oxygen evolution and the role of bicarbonate in this process, outputs of the past year have not yet been formally disseminated to the public as in the previous year. The results have been discussed at length with colleagues in the field by way of email, phone, and in-person contacts. However, to inform and benefit a wider audience will require additional findings just now coming to light.
Impacts
In the recent past we published a model of photosynthetic oxygen evolution postulating a role for bicarbonate ions and their oxidized form, peroxydicarbonic acid, as chemical intermediates. One requirement of this model is that photosystem II (PSII) must be able to catalytically hydrate CO2. That is, it must act as the enzyme carbonic anhydrase (CA). It happens that there are two sources of CA activity in the PS II complex. Work in the past year resulted in an unexpected and serendipitous discovery related to our assay for determining CA activity. Often, for no apparent reason, our results were inconsistent, motivating a protracted search for a hidden experimental variable. To measure CA activity, our samples are stirred with a magnetic stirrer, like those used in many types of assays. Although it is not commonly recognized, a moving electric field can affect the physical and chemical properties of water. This has been reported in a few journals and on the Internet, but
the theoretical basis for the phenomenon has not yet been established. Hence, knowledge and acceptance of this effect is not general. We found that our inconsistent results were a function of the strength, distance, and exposure time of the magnetic field to our samples. The effect is not large, and requires a sensitive system like ours to detect. In most systems that employ magnetic stirring, it will go unnoticed. However, in a few instances, e.g. in sensitive assays in which water is a reactant, the effect of a rotating magnetic field can be significant. Others should be informed of this subtle variable. We are now able to control the magnetic field effects by keeping all samples in a MuMetal box that shields against these fields. Consequently, we have returned to the planned experiments studying PS II CA. While typical CA enzymes are inhibited by monovalent anions such as formate, the innermost PS II CA shows a complex relationship with this anion. At low pH and low formate
concentrations, CA activity actually increases, while higher concentrations inhibit as expected. To explain these results, we refer to information we gathered years ago. There is a pool of CO2/HCO3- bound to PS II. This pool, though tightly bound, can be removed at low pH and with the addition of formate. We therefore propose that the innermost PS II CA acts on this pool of tightly bound CO2/HCO3-. However, in vivo, the product of catalysis, HCO3-, is not released into the medium but is retained to be used in oxygen evolution. If this is the case, little CA activity will be measured because the assay requires continuous release of HCO3- into the medium. At low pH, however, addition of formate will initially cause the release of HCO3- into the medium (as experimentally observed) thus resulting in an apparent increase in CA activity. High concentrations of formate, on the other hand, will cause the active site to be permanently occupied by formate. This will prevent the binding of
substrate CO2 and thus inhibit CA activity. The presence of CA activity acting on a tightly bound pool of HCO3- on PS II provides additional support for the model of oxygen evolution we have proposed.
Publications
- No publications reported this period
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Progress 01/01/06 to 12/31/06
Outputs
Our study of the primary chemical reactions of photosynthesis has seen further developments. The study focuses on the mechanism whereby oxygen is evolved in plants by the decomposition of water molecules. This takes place in a protein/chlorophyll complex (photosystem II, or PSII) in chloroplast inner membranes. It is in PSII that absorbed light is used to extract electrons from water for the purpose of synthesizing organic molecules. In the process, molecular oxygen is given off. We have pursued the role of necessary cofactors in this process, particularly bicarbonate ions (HCO3-) but also Cl-, Ca2+, and Mn. We have found in the past that PSII has carbonic anhydrase (CA) activity, the ability to catalytically produce HCO3- from H2O and dissolved CO2. Our work has concentrated on locating and characterizing this activity. We found that there are actually two sources of CA activity associated with PSII. One is on an extrinsic protein (OEC33) attached to the PSII complex
and removable by washing the membranes with 1 M CaCl2. When this CA is removed, another source of activity remains, intrinsic PSII CA. To characterize these two forms of CA, we compared the effects of Cl-, Mn2+, Ca2+, and pH on extrinsic and intrinsic PSII CA activity . We found that extrinsic CA activity, located on the OEC33 protein, was optimum at about 30 mM Cl-, and strongly inhibited above this concentration. This enzyme is activated by Mn2+ and stimulated somewhat by Ca2+. The OEC33 showed dehydration activity that is optimum at pH 6 or below. In contrast, intrinsic CA activity found in the PSII complex after removal of extrinsic proteins was stimulated by Cl- up to 0.4 M. Ca2+ appears to be the required cofactor, which implies that the location of the intrinsic CA activity is in the immediate vicinity of the CaMn4 complex that is directly responsible for oxygen evolution. Up to now, intrinsic CA has shown only hydration activity that is nearly pH independent. Based on the very
different characteristics of the two forms of PSII CA activity we suggest that they may have different functions in PSII as well. We now have a manuscript accepted that details a new chemical model for oxygen evolution. The model is unique in postulating that the immediate source of photosynthetically-evolved oxygen is water bound to CO2 to form HCO3-. The bicarbonate donates an electron to PSII and forms the bicarbonate radical. When two such radicals combine, they form the unstable peroxydicarbonic acid (Podca). Upon further oxidation, this proposed intermediate evolves oxygen. We were able to synthesize Podca electrochemically and stabilize it at low temperature. In the presence of Mn2+ or Co2+ Podca was quickly broken down with release of O2. In the presence of Ca-washed photosystem II-enriched membranes lacking extrinsic proteins, Podca was decomposed with the release of O2, but only under conditions favoring photosynthetic electron flow (light plus an electron accepter).
Although far from proving our hypothesis, these results provide indirect evidence that Podca could act as the immediate precursor of photosynthetically evolved oxygen. We continue to test this model.
Impacts
Uncovering the mechanism of oxygen evolution remains the great challenge in the field of photosynthesis. For the past half-century, hundreds of researchers around the world have attempted to "pull the sword from the rock." The reward for doing so is manifest. To convert light energy into vast amounts of useful chemical forms artificially, such as hydrogen gas, is the continued dream of photochemists and energy engineers. However, for artificial systems to use abundant solar energy to produce hydrogen gas from water, knowledge of how water splitting occurs in green plants will be invaluable, if not absolutely essential. The research described here is a reasonable approach to the goal of understanding the enigma of oxygen evolution. With this knowledge there is hope of solving some of our energy problems.
Publications
- Lu Y-K and Stemler A.J. (2006) Differing responses of the two forms of photosystem II carbonic anhydrase to chloride, cations and pH. Biochimica et Biophysica Acta, in press.
- Castelfranco P.A., Lu Y-K and Stemler A.J (2006) Hypothesis: The peroxydicarbonic acid cycle in photosynthetic oxygen evolution. Photosynthesis Research, in press.
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Progress 01/01/05 to 12/31/05
Outputs
We continue to make progress in our study of the initial chemical reactions of photosynthesis. These reactions take place inside a protein pigment complex (photosystem II) located in chloroplast thylakoid membranes. Here, light energy is used to split two water molecules with the release of molecular oxygen. Yet, as fundamental as this process is to life on earth, detailed knowledge of the chemical mechanism has escaped researchers around the world for decades. What is known is that besides substrate water, several inorganic cofactors are required, manganese and calcium cations, and chloride and bicarbonate anions. We have focused on understanding the role of the last of these, bicarbonate. The study involves two approaches. The first is to investigate the ability of photosystem II to catalytically produce bicarbonate from dissolved carbon dioxide and water, a reaction normally done by the enzyme carbonic anhydrase. We have determined in the past that the photosystem
II complex has two sources of carbonic anhydrase activity. As described in our recently published work, we located one source of activity on the extrinsic 33 kDa protein attached to the lumenal face of the photosystem II complex. This carbonic anhydrase is very unusual in requiring manganese as a metal cofactor instead of zinc. Like the 33 kDa protein itself, the activity is heat stable, and survives a 15 minute treatment at 90 degrees C. Strangely, the 33 kDa protein shows no primary sequence homology to any of three families of carbonic anhydrase described in the databases. We continue to characterize this, as well as the second source of carbonic anhydrase activity that remains on photosystem II after the 33 kDa extrinsic protein is removed. Our second approach to understanding the role of bicarbonate in oxygen evolution was to begin to test the hypothesis put forward by the late Helmut Metzner over twenty years ago. He proposed that bicarbonate was the immediate electron donor to
photosystem II, and not water directly. Accordingly, two bicarbonate radicals thus formed condense to form a molecule of peroxydicarbonic acid, and two of these could dismutate to release molecular oxygen. Another product, carbon dioxide, would then be catalytically rehydrated with endogenous carbonic anhydrase to continue the cycle. We were able to synthesize peroxydicarbonic acid electrochemically and, although it is unstable at room temperature, keeps indefinitely at -80 degrees C. We found that this compound, when thawed, reacts with manganese in solution to yield molecular oxygen. Moreover, when peroxydicarbonic acid is given to illuminated photosystem II membranes, oxygen evolution is enhanced. This means that peroxydicarbonic acid can act as an electron donor to photosystem II and as a source of evolved oxygen. This is in accordance with the Metzner model, although proof remains elusive. We are now working to improve our method for producing and stabilizing peroxydicarbonic
acid to repeat and extend our present results. Meanwhile, we have developed, and are beginning to test, a detailed chemical model of oxygen evolution based on the Metzner model.
Impacts
To convert light energy into useful chemical forms artificially and in vast amounts, is the continued dream of photochemists and energy engineers. The green plant does this naturally by the process of photosynthesis, but nature has kept the initial chemistry of water-splitting and concomitant oxygen evolution a secret. Until that secret is revealed, we will be unable to apply these principles to solving at least some of our energy problems. The research described is a reasonable approach to uncovering the fundamental chemistry of water splitting. Given eons of natural selection to optimize photosynthesis, it is unlikely that this knowledge will improve plant productivity. However, for artificial systems to use abundant solar energy to produce hydrogen gas from water, knowledge of how water splitting occurs in green plants will be invaluable, if not absolutely essential.
Publications
- Lu Y-K., Theg S.M. and Stemler A.J. (2005) Carbonic anhydrase activity of the photosystem II OEC-33 protein from pea. Plant Cell Physiology 46: in press.
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Progress 01/01/04 to 12/31/04
Outputs
Photosynthesis in plants is a multi-phase process that begins with the absorption of light by a complex of proteins and chlorophyll molecules, photosystem II, embedded in the thylakoid membranes of chloroplasts. The energy is transferred to a special pair of chlorophyll a molecules where photochemistry is initiated. One of the first stable chemical products of these initial steps is molecular oxygen, derived from the splitting of water molecules. It is this initial chemistry, the oxidation of water molecules that has captured the interest of hundreds of researchers worldwide. We continue to study the cofacters, such as manganese, and enzymatic activities associated with oxygen evolution to eventually unlock this mystery. One cofacter, bicarbonate ions, has especially drawn our attention. We have shown that the oxygen-evolving complex has the ability to catalytically produce this ion from carbon dioxide and water. That is, it has the enzymatic activity of carbonic
anhydrase. The requirement for bicarbonate, and the presence of carbonic anhydrase activity caused us to reinvestigate an obscure model of oxygen evolution proposed by Helmut Metzner and Werner Kreutz more than twenty years ago. These workers suggested that oxygen is evolved from peroxydicarbonic acid (Podca), a compound formed from the joining of two bicarbonate radicals produced in the light by photosystem II. However, Metzner and Kreutz were never able to provide convincing evidence in support of their model. Unlike these workers, we have been able to electrolytically synthesize Podca, and are presently studying its characteristics. We have found that it is very unstable at room temperature, but can be kept indefinitely at - 80 degrees Celsius. When Podca breaks down spontaneously at room temperature, hydrogen peroxide is released. In the presence of manganese ions, however, molecular oxygen is given off instead. However, in the presence of manganese, some of the Podca oxidizes the
organic buffers used to maintain pH. This indicates the powerful oxidizing ability of this compound. We then tested the ability of Podca to release oxygen in the presence of photosystem II membranes and light. We found that Podca is indeed an effective electron donor to photosystem II and molecular oxygen is released as a result, thereby mimicking the natural process. Hydrogen peroxide, in contrast, did not yield oxygen and therefore it was not the breakdown product of Podca that was entering the reaction, but Podca itself. This circumstantial evidence does not by any means prove that the Metzner-Kreutz hypothesis is correct. However, it does indicate that the model should be tested further. We are now in the process of writing up our current results, and planning more definitive experiments.
Impacts
The chemical mechanism by which plants replenish the atmosphere with oxygen has been described as the "Holy Grail" of photosynthesis. For over half a century, thousands of researchers have devoted countless hours and innumerable resources to this quest. Dozens of models fill the literature but in every case, the evidence is weak. We believe that the model proposed by Metzner and Kreutz currently has more supporting evidence than any other and therefore deserves close examination. If the model proves correct, the longest-standing mystery in photosynthesis will be solved and we can go on to create artificial photosynthesis to convert solar energy into stable fuels and other useful carbon compounds.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
In plants, photosynthesis begins with the light-driven decomposition of water and the release of molecular oxygen in a protein-pigment complex located in chloroplast thylakoid membranes. This complex, known as photosystem II (PSII) is the initial site where light energy from the sun is converted to stable chemical forms that can then be used to maintain not only plant life, but nearly all biological systems on earth. We continue to study the workings of PSII with the expectation of eventually uncovering the chemical mechanism of oxygen evolution. This past year we have concentrated on characterizing the enzymatic activity that we discovered was associated a particular extrinsic protein (OEC33) attached to PSII on the inside surface of thylakoids. This protein has carbonic anhydrase (CA) activity, and can catalyze the reversible hydration of CO2 to form bicarbonate, HCO3-. This anion is known to have some, so far unspecified, role in oxygen evolution. We propose that
the OEC33 can supply PSII with bicarbonate to fulfill this requirement. We have found that the OEC33 has unique properties compared to all other CAs described. The primary structure bears no primary structure homology to any other CA. The metal cofactor required for activity is manganese, not zinc as is used in every other CA. It is thermally stable, and retains enzymatic activity after treatment at 90˚C for 15 minutes. Most unusual, the activity is controlled by ultra-violet (UV) light. In the dark, the enzyme is only capable of catalyzing the equilibrium, carbonic acid (H2CO3) =C HCO3- and H+. After treatment with low levels of UV light, the enzyme then catalyzes exclusively the equilibrium, CO2 + H2O C= H2CO3. We have demonstrated these unique characteristics in vitro with enzyme that was expressed in E. coli. The actual in vivo function of the OEC33 remains uncertain, but we found evidence that its carbonic anhydrase activity is important for oxygen evolution. When the CA
activity is inhibited by the specific inhibitors acetazolamide and ethoxzolamide, oxygen evolution is likewise inhibited. This evidence supports our hypothesis that the function of the OEC33 is to supply HCO3- to the PSII complex. We are now characterizing the 'dark' and 'UV-light' forms of the OEC33 described above. It is probable that the high-energy light causes an oxidation of the enzyme. If so, the light effect should be induced chemically at the appropriate oxidation-reduction range. We have also completed reconstitution experiments in which the OEC33 was first removed from PSII by washing membranes with 1 M CaCl2. This removed both membrane bound CA activity and most of the oxygen-evolution. Both activities were restored by adding back E. coli- expressed OEC33. This confirmed that the OEC33 is the site of CA activity and demonstrates once again the correlation between the CA activity and oxygen evolution. Once the carbonic anhydrase activity of PSII is fully characterized, we
will begin testing the published chemical models that are based on the idea that bicarbonate ions are the immediate source of photosynthetically-evolved oxygen.
Impacts
For over half a century several thousand researchers around the world have sought to describe the chemical mechanism by which plants make oxygen to replenish the atmosphere. Detailed knowledge of how nature accomplishes this amazing feat is needed before we can hope to reproduce this form of solar energy capture. The work described represents the most promising approach to achieve this goal.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
The first steps in photosynthesis result in the decomposition of water and the release of molecular oxygen. Cofactors for this process include manganese, calcium, chloride and bicarbonate. The exact roles played by these ions remain highly speculative despite much effort over many years. We continue to study the role of bicarbonate ions and the enzyme that produces it, carbonic anhydrase (CA), in the mechanism of oxygen evolution. We discovered in the past that there are two sources of CA activity closely associated with photosystem II and the oxygen-evolving machinery. In the past year we have further characterized these sources of CA activity. We have obtained additional evidence that one CA is the 33 kDa extrinsic protein on PSII. This CA is unique in requiring manganese for activity. Another source of CA activity is more firmly attached to the membrane and requires calcium. The concentration dependence of the tightly-bound CA for calcium is the same as for oxygen
evolution. When extrinsic proteins are removed from PSII, oxygen evolution becomes dependent on very high concentrations of chloride. This is also true for the tightly-bound CA activity. The strong correlation in the need for similar concentrations of calcium and chloride between oxygen evolution and tightly-bound CA allows the suggestion that these two cofactors may indirectly stimulate oxygen evolution by maintaining the supply of bicarbonate from tightly-bound CA. Such roles for calcium and chloride have never before been proposed. The 33 kDa activity is blocked by acetazolamide and ethoxzolamide. Both compounds are classical inhibitors of CA. The tightly bound CA form, in contrast, is immune to these inhibitors. In the absence of the 33 kDa protein, oxygen evolution is likewise immune to CA inhibitors. However, in the presence of the 33 kDa protein, oxygen evolution is reduced by CA inhibitors. These results imply, for the first time, that the 33 kDa protein has a strong
regulatory role in oxygen evolution. We have also found that the two forms of CA also have different enzymatic functions. The tightly bound CA appears to catalyze only the hydration reaction, converting carbon dioxide to bicarbonate. The 33 kDa form does only the reverse, dehydration reaction. This means, that by varying the substrate and conditions, the two enzymes can be assayed independently, even when both forms of CA are present in the PSII complex. It also implies that the two enzymes have very different roles in oxygen evolution, despite their close proximity. We propose that the tightly-bound CA supplies bicarbonate to the oxygen-evolving mechanism and that the bicarbonate may act as the immediate source of evolved oxygen rather than water directly. This hypothesis has been in the literature for some time, but had little supporting evidence. The foregoing results suggest that it is time to reevaluate this possible model for oxygen evolution. We are now in the process of
testing this idea further. The role of the 33 kDa CA activity is much less obvious and therefore more intriguing. More detailed characterization, now in progress, may provide the necessary clues.
Impacts
Photosynthesis is responsible for the food we consume and the air we breathe. It is critical to understand all aspects of this process to ensure continued human and environmental well being. The work discussed provides insight into the initial chemical steps of photosynthesis, the least understood aspect of the process. Knowledge of how nature accomplishes this feat is the first step in engineering systems for converting light energy into stable chemical forms.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
The study of the mechanism of photosynthetic oxygen evolution in plants has continued. In particular, we have concentrated on the role of the bicarbonate ion, an important but little understood cofactor in the process. We have shown that the photosystem II complex that evolves oxygen also has two sources of carbonic anhydrase activity and can catalytically convert carbon dioxide to bicarbonate. We have now identified one source as the 33 kDa extrinsic protein associated with the oxygen-evolving complex. For activity, this extrinsic carbonic anhydrase (CAext) requires chloride ions, another known cofactor in oxygen evolution. The CAext activity is also stimulated by manganese. These characteristics are unlike any carbonic anhydrase described in the literature. Another unique feature of this enzyme is its complete lack of primary amino acid sequence homology to any member of the three families of CA documented. The CAext therefore represents a new class of carbonic
anhydrase. We also completed a study of the location of the two forms of CA in maize. Since maize is a C4 plant, it has two distinct types of chloroplasts in leaves. One type is in the mesophyll cells, the other in bundle sheath cells. Characteristic of the bundle sheath chloroplast is a lack of photosystem II and an inability to evolve oxygen. We determined that these chloroplasts contain neither form of CA. In contrast, mesophyll chloroplasts contain photosystem II, evolve oxygen, and exhibit both forms of CA activity. These results show that the two carbonic anhydrases correlate to the presence of the oxygen-evolving machinery and strongly imply some functional role for these enzymes, and bicarbonate, in oxygen evolution. We continue to characterize the two forms of CA on photosystem II and we plan to test the ideas we, and others, have published on the role of bicarbonate in the chemistry of oxygen evolution.
Impacts
The mechanism of water decomposition and the release of molecular oxygen remains the greatest mystery in the field of photosynthesis. The work in progress is providing vital clues to the chemistry involved in this process. Since photosynthesis is responsible for all the food we consume and the oxygen we breath, it is crucial that we understand how these first steps in photosynthesis take place.
Publications
- Stemler, A.J. and Lu, Y-K PSII carbonic anhydrase activity and the bicarbonate effect. Proceedings of the XIIth International Congress on Photosynthesis, Brisbane, Australia, July17-23, 2001.
- Lu, Y-K. and Stemler, A Extrinsic photosystem II carbonic anhydrase in maize mesophyll chloroplasts. Plant Physiology 128:1-7, 2002 (in press)
- Stemler A.J. The bicarbonate effect, oxygen evolution, and the shadow of Otto Warburg. Photosynthesis Research 2002 (in press)
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Progress 01/01/00 to 12/31/00
Outputs
In studying the mechanism of oxygen evolution that takes place in chloroplasts during photosynthesis, we focussed on the role of carbonic anhydrase (CA) in the process. This enzyme is capable of hydrating CO2 in water to form bicarbonate ions. Bicarbonate is a recognized cofactor in oxygen evolution and is suggested to function as a chemical substrate. We discovered that chloroplast membranes have two distinct sources of CA activity. One source is a protein in the thylakoid lumen that can be removed and characterized in vitro. Another source of CA activity is tightly bound to the membrane and appears to emanate from the oxygen-evolving mechanism itself. The two sources of CA activity have different ion requirements and different pH responses. While lumenal CA is inhibited by chloride, the tightly bound form actually requires 0.4 M chloride for optimum rates. The bound form also requires added calcium (5 mM). In its requirement for chloride and calcium, the bound form
of CA exactly parallels oxygen evolution which also requires these anions. Unlike the lumenal CA, (and all other CA reported) the tightly bound form can hydrate CO2 at pH below 6. This is significant because the oxygen-evolving mechanism operates at a pH near 5. If bicarbonate is required for oxygen evolution, the mechanism to supply it, i.e. carbonic anhydrase, must operate at this low pH. Thus it appears that the mechanism for producing bicarbonate is present, and supports the hypothesis that bicarbonate could be the chemical substrate for oxygen evolution.
Impacts
Research into the role of carbonic anhydrase in photosynthetic oxygen evolution will allow us to understand the primary photochemical steps in photosynthesis. Since this process is responsible for all of our food and the oxygen we breathe, it is imperative that we know how to protect and maximize photosynthesis in our agricultural plants in particular. Knowledge of this process will help us develop artificial means of storing light energy in chemical form.
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
Further progress was made characterizing the carbonic anhydrase activity associated with photosystem II in maize chloroplast membranes. Carbonic anhydrase activity is the ability to reversibly hydrate CO2 dissolved in water to form bicarbonate (HCO3-). Photosystem II is a large multi-protein complex that initiates the light-driven steps of photosynthesis. It is able to extract electrons from water and releases molecular oxygen as a by-product. The role of carbonic anhydrase in this process is speculative. We hypothesize, based on the known requirement of photosystem II for bicarbonate, that this anion plays some role in the chemistry of oxygen evolution. If so, then the ability of the photosystem II complex to catalyze the production of bicarbonate (i.e., the presence of carbonic anhydrase activity) would be explained. As a result of the past year's work, we are now aware that there are two sources of carbonic anhydrase activity near photosystem II. One source is a
loosely-bound, approximately 30 k Dalton protein located in the lumen of the chloroplast membranes. Resembling the type of enzyme recently described in the alga Chlamydomonas. It can be solubilized from photosystem II membranes by washing in 0.2 M salt. However, there exists another source of carbonic anhydrase activity in the membranes. This source is very tightly bound and seems to be associated with the oxygen-evolving mechanism. We are now faced with the task of characterizing these two sources of activity separately, and determining their physiological functions.
Impacts
Research into the role of carbonic anhydrase in photosynthetic oxygen evolution will allow us to understand the primary photochemical steps in photosynthesis. Since this process is responsible for all of our food and the oxygen we breathe, it is imperative that we know how to protect and maximize photosynthesis in our agricultural plants in particular. Knowledge of this process will help us develop artificial means of storing light energy in chemical form.
Publications
- No publications reported this period
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Progress 01/01/98 to 12/31/98
Outputs
Research was conducted on the early chemical events in photosystem II. This protein-chlorophyll complex is located in chloroplast membranes and is responsible for the conversion of light energy into chemical form. In the process, water molecules are split and molecular oxygen is released. Cofactors in this process include chloride, bicarbonate, and manganese ions. To make bicarbonate, the photosystem II complex has the ability to hydrate CO2 catalytically, and in this respect acts like the enzyme carbonic anhydrase. The exact role of bicarbonate in oxygen evolution is not known, nor the reason for carbonic anhydrase activity. Of the numerous carbonic anhydrases described in the literature, all require zinc as the metal cofactor. Photosystem II, in contrast, is not known to contain zinc, nor is this metal known to be a requirement for oxygen evolution. We thus explored the possibility that the metal cation associated with photosystem II carbonic anhydrase activity was
manganese. Attempts were made to remove and then restore manganese to the photosystem II complex while both oxygen evolution and carbonic anhydrase activity were monitored. Upon removal of some manganese, oxygen evolution was eliminated, while carbonic anhydrase activity remained. It would appear that while manganese is necessary for oxygen evolution, it is not required for carbonic anhydrase activity. However, since each photosystem II complex contains four manganese ions, we must measure the amount of metal ions remaining after our removal treatment to make sure all was removed. Until we do this, our results remain ambiguous. These experiments are continuing.
Impacts
(N/A)
Publications
- Stemler, A.J. Photosystem II carbonic anhydrase activity depends on and Ca++ Proceedings of the XIth International Congress on Photosynthesis, August 17-22, Budapest, Hungary, in press.
- Stemler, A.J. Bicarbonate and photosynthetic oxygen evolution: An unwelcome legacy of Otto Warburg. Indian J Exptl Biol 36: 841-848,
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Progress 01/01/98 to 12/01/98
Outputs
Research was conducted on the early chemical events in photosystem II. This protein-chlorophyll complex is located in chloroplast membranes and is responsible for the conversion of light energy into chemical form. In the process, water molecules are split and molecular oxygen is released. Cofactors in this process include chloride, bicarbonate, and manganese ions. To make bicarbonate, the photosystem II complex has the ability to hydrate CO2 catalytically, and in this respect acts like the enzyme carbonic anhydrase. The exact role of bicarbonate in oxygen evolution is not known, nor the reason for carbonic anhydrase activity. Of the numerous carbonic anhydrases described in the literature, all require zinc as the metal cofactor. Photosystem II, in contrast, is not known to contain zinc, nor is this metal known to be a requirement for oxygen evolution. We thus explored the possibility that the metal cation associated with photosystem II carbonic anhydrase activity was
manganese. Attempts were made to remove and then restore manganese to the photosystem II complex while both oxygen evolution and carbonic anhydrase activity were monitored. Upon removal of some manganese, oxygen evolution was eliminated, while carbonic anhydrase activity remained. It would appear that while manganese is necessary for oxygen evolution, it is not required for carbonic anhydrase activity. However, since each photosystem II complex contains four manganese ions, we must measure the amount of metal ions remaining after o.
Impacts
(N/A)
Publications
- STEMLER, A.J. Photosystem II carbonic anhydrase activity depends on Cl-and Ca++ Proceedings of the XIth International Congress on Photosynthesis, August 17-22, Budapest, Hungary, in press.
- STEMLER, A.J. Bicarbonate and photosynthetic oxygen evolution: An unwelcome legacy of Otto Warburg. Indian J Exptl Biol 36: 841-848,
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Progress 01/01/97 to 12/01/97
Outputs
Research focused on the initial stages of photosynthesis, particularly the mechanism whereby plants release oxygen from water. This chemical reaction, the beginning of food production by green plants, remains little understood. One problem is that the oxidation state of the population of oxygen-evolving protein complexes is heterogeneous. Working with a French colleague, Jerome Lavergne, I showed that formate ions could allow the reduction of the entire population of oxygen-evolving complexes to a single uniform "ground" state. The properties of this state can now be studied as can the homogeneous higher oxidation states of the complex produced by light flashes, leading to a more complete understanding of the chemistry of oxgyen evolution. In addition, we continued to characterize carbonic anhydrase activity associated with chloroplast membranes. This activity, the reversible hydration of dissolved CO2 is postulated to play some role in oxygen evolution. We compared
the chloride concentration dependence of oxygen evolution and membrane carbonic anhydrase. In whole membranes, both activities saturate at less than 20 mM chloride. In membranes treated to concentrate the oxygen-evolving complexes and washed with salt to remove extrinsic proteins, the chloride concentration optimum for both activities shifts to 30 mM while higher concentrations inhibit both. These parallel responses to chloride suggest that carbonic anhydrase activity may indeed be associated with the oxygen-evolving mechanism.
Impacts
(N/A)
Publications
- STEMLER, A.J. 1996. The Case for Chloroplast Thylakoid Carbonic Anhydrase. Physiologia Plantarum. In press.
- STEMLER, A.J. 1996. Evidence that Formate Destabilizes the S1 State of the Oxygen-Evolving Mechanism. Photosynthesis Research. Accept for
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Progress 01/01/96 to 12/30/96
Outputs
Research focused on the initial stages of photosynthesis, particularly the mechanism whereby plants release oxygen from water. This chemical reaction, the beginning of food production by green plants, remains little understood. One problem is that the oxidation state of the population of oxygen-evolving protein complexes is heterogeneous. Working with a French colleague, Jerome Lavergne, I showed that format ions could allow the reduction of the entire population of oxygen-evolving complexes to a single uniform "ground" state. The properties of this state can now be studied as can the homogeneous higher oxidation states of the complex produced by light flashes, leading to a more complete understanding of the chemistry of oxygen evolution. In addition, we continued to characterize carbonic anhydrase activity associated with chloroplast membranes. This activity, the reversible hydration of dissolved CO2, is postulated to play some role in oxygen evolution. We compared
the chloride concentration dependence of oxygen evolution and membrane carbonic anhydrase. In whole membranes, both activities saturate at less than 10 mM chloride. In membranes treated to concentrate the oxygen-evolving complexes and washed with salt to remove extrinsic proteins, the chloride concentration optimum for both activities shifts to 30 mM while higher concentrations inhibit both. These parallel responses to chloride suggest that carbonic anhydrase activity may indeed be associated with the oxygen-evolving mechanism.
Impacts
(N/A)
Publications
- STEMLER, A. J. 1996. The Case for Chloroplast Thylakoid Carbonic Anhydrase, Physiologia Plantarum. In press.
- STEMLER, A.J. 1996 Evidence that Formate Destabilizes the S1 State of the Oxygen-Evolving Mechanism. Photosynthesis Research. Accept for pub.
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