American Scientist

Almost four billion years ago, some unicellular organisms developed the ability to use light from the sun as a plentiful energy source. By combining light energy and available sources of chemicals, these organisms evolved into the first photosynthetic species.

Most people associate the term "photosynthesis" with the ability of green plants to use energy from light to power the manufacture of sugars and other organic molecules. However, what is sometimes forgotten is that photosynthesis also supplies all of the molecular oxygen, or dioxygen (O₂), we breathe. For this reason alone, it is probably not surprising that understanding O₂ production by plants and other photosynthetic organisms has long been a focus of research in photosynthesis. The surprising part is that we still do not know exactly how plants generate O₂, even though its role in photosynthesis was discovered more than 200 years ago.

To be sure, much has been learned in that time. Scientists have found that chloroplasts are the subcellular organelle in which photosynthesis takes place. They have even discovered many of the proteins and inorganic molecules that carry out the energy conversion. And yet, when it comes to the very last step, the one where dioxygen is actually formed, scientists are still working out the precise molecular mechanisms. At the core of their studies is a manganese-containing complex embedded in the chloroplast membranes, called the oxygen-evolving complex. The structure of this complex is still largely unknown, but some of its features can be deduced and modeled. These deductions have led, in turn, to some interesting proposals about the way plants produce oxygen.

The Bell Jar

In the late 18th century, Joseph Priestley, an English Unitarian minister, used glass bell jars to trap various gaseous components of air. In 1772, he devised an experiment to determine whether plants and mice were similarly affected by air that contained carbon dioxide (CO₂) but not O₂. To his surprise, he found that a mouse quickly died in the bell jar lacking O₂, whereas a sprig of mint continued to live. Perhaps more important, when a mouse was placed in a jar from which mint had just been removed, the mouse remained alive. The plant had changed the CO₂ atmosphere by producing O₂. It was another seven years before the Dutch physician Jan Ingen-Housz showed that plants need to be placed in direct sunlight in order for O₂ to be generated. Twenty-five years after Ingen-Housz's discovery, Swiss scholar Théodore de Saussure was studying the source of matter in growing plants. He reported that the combined weights of the O₂ produced by plants and the organic matter contained in plants was too large to be derived from CO₂ alone. Therefore, he reasoned that water (H₂O), the only other material he had added, must also be absorbed by plants. Combining these earlier observations, in 1842 the German physiologist Julius von Mayer surmised that plants convert light energy from the sun into chemical energy through the process now called photosynthesis.

Putting all of this information together, scientists concluded that plant photosynthesis is a process that uses light energy to convert CO₂ and H₂O into carbohydrates and dioxygen. The overall reaction for plant photosynthesis is shown in the equation in Figure 2.

One of the most enduring questions, however, has been the exact source for the oxygen. During the first few decades of the 20th century, it was believed that CO₂ and water combined to produce carbohydrates and that the oxygen atoms in O₂ were derived directly from CO₂. In the early 1930s, Cornelis van Niel of Stanford University was studying anaerobic bacteria that use hydrogen sulfide (H₂S) instead of H₂O in photosynthesis and found that the bacteria generate sulfur (S₈) as a byproduct. Along with other studies of plant and bacterial photosynthesis, van Niel's result led him to propose that photosynthesis consists of two separate reactions. The first of these reactions is oxidation of a compound with the general formula H₂A (for example, H₂S or H₂O) with the concomitant generation of protons (H⁺) and electrons (e^–), as seen in the first equation of Figure 3. This reaction requires light to proceed and is now referred to as the light reaction. The second reaction uses the protons and electrons generated by the first reaction to reduce CO₂ and produce carbohydrates and water (second equation of Figure 3). Because CO₂ reduction does not depend directly on light, it is called the dark reaction.

Dividing photosynthesis into two elementary steps helps classify photosynthetic organisms based on the source of electrons (H₂A) for the light reaction. The anaerobic bacteria studied by van Niel belong to a larger order of photosynthetic bacteria, Rhodospirillales, which use reduced sulfur compounds like hydrogen sulfide, sulfur or other sulfide compounds (S^2–), or hydrogen (H₂) in the light reaction. Plants, algae and cyanobacteria (previously called blue-green algae) use only H₂O as the electron donor in the light reaction and generate O₂ as the light-reaction product.

Light Harvesting

In order to convert solar energy into chemical energy, photosynthetic organisms must be able to harness light energy. Typically, plants "harvest" light by using molecules that absorb light energy from different regions of the solar spectrum. Because these light-harvesting compounds absorb only some energies of visible light and reflect others, they are brightly colored and are referred to as chromophores or pigment molecules.

The primary chromophore in plants is chlorophyll. It is also what makes plants green (Figure 6, top). Chlorophylls are found inside the membranes of organelles called chloroplasts. Probably descendants of cyanobacteria, plant chloroplasts contain all of the proteins and pigments required for the synthesis of carbohydrates. They vary in size, but are typically ellipsoids about 5 micrometers long. In addition to chlorophyll, the chloroplasts contain all of the proteins and auxiliary molecules of the chemical reactions of photosynthesis.

There are several different types of chlorophyll, all of which possess two general structural features: a nearly planar core that contains a magnesium ion in the center and a long tail to anchor the chlorophyll molecule in its host environment. Depending on other structural elements, plant chlorophylls are designated "a" or "b." Chlorophylls in bacteria (or bacteriochlorophylls) differ from those found in plants, although they also exist in a and b forms. Other chromophores involved in photosynthesis include the linear carotenoids, a familiar example of which is β-carotene, the pigment that makes carrots orange.

The pigment molecules in photosynthetic organisms vary, but all species exploit the ability of the various chromophores to absorb sunlight of particular energies and wavelengths. Therefore, when a plant uses carotenoids in combination with chlorophylls a and b, almost the entire spectrum of light coming from the sun can be absorbed for use in photosynthesis (Figure 6, bottom).

Even though chlorophyll absorbs light in two different regions of the solar spectrum (wavelengths of 400–500 nanometers and 600–700 nanometers), all of the absorbed light is equally useful in photosynthesis because of the unique electronic properties of chlorophyll. Another advantage of using chlorophylls to harvest light is that chlorophyll molecules can transfer energy from one to another with very little energy loss. Therefore, when a single chlorophyll molecule absorbs light energy, it can transfer that energy to a neighboring chlorophyll molecule. The second chlorophyll can then pass the absorbed energy on to a third chlorophyll molecule. In this way, light energy can be relayed to a particular location in the photosynthetic organism (Figure 7).

Once harvested, the light energy is ultimately relayed to the reaction center, where the chemical reactions of photosynthesis take place. Photosynthetic reaction centers are multiple-protein assemblies that bind other small molecules, called cofactors, required for photosynthesis. They are further characterized by their location in membranous regions of the chloroplasts.

Two types of membranes make up a chloroplast: an outer encapsulating membrane and an inner membrane called the thylakoid membrane (Figure 4). The thylakoid membrane is a long, continuous membrane that folds on itself to produce two types of secondary structures called grana and stroma lamellae. The grana are regions of thylakoid membrane that are folded into stacks, and the stroma lamellae are unfolded lengths of thylakoid membrane that connect the grana.

Chlorophyll and all of the protein complexes required for the light reaction of plant photosynthesis are found in the thylakoid membranes. This includes the photosynthetic reaction centers, Photosystem I (PSI) and Photosystem II (PSII), in addition to a protein complex that synthesizes ATP (a molecule that stores chemical energy) and the cytochrome b₆/f complex, which is chiefly involved in shuttling protons and electrons from one side of the thylakoid membrane to the other (Figure 5). Because PSI and PSII are intimately involved in the light reaction in plants, they are major targets of photosynthesis research. PSI uses the electrons received from PSII to generate the strong reductant, NADPH, which is important in fixing carbon dioxide.

PSII is of particular interest because it is the reaction center directly responsible for the oxidation of H₂O to O₂. Experiments involving PSI and PSII are simplified when discrete PSI and PSII units can be selectively isolated. This is accomplished by taking advantage of the fact that stroma lamellae contain mostly PSI, whereas the grana contain predominantly PSII.

Charge Separation, Electron Transfer

When light hits a plant leaf, chlorophylls in the chloroplast thylakoid membrane harvest the light energy and deliver it to the PSII reaction center. What happens to the light energy when it reaches PSII, and how does the light energy get converted to chemical energy? Like chlorophylls in the thylakoid membrane, the chlorophyll molecules in PSII accept the light energy transferred to them. However, one chlorophyll species in PSII differs from other chlorophylls in two respects. First, the chlorophyll species in PSII "traps" light energy as soon as it arrives by initiating a charge-separation reaction, prohibiting escape of the light energy to surrounding chlorophylls. Second, the chlorophyll species in PSII is not a spatially isolated chlorophyll molecule like those found in other regions of the thylakoid membrane. Instead, it is composed of two adjacent and identical chlorophyll molecules. For this reason, it is called a special pair.

The special pair of chlorophylls in PSII traps light energy in order to turn it into chemical energy. The first step through which photosynthetic organisms convert light energy to chemical energy is called photoexcitation (Figure 9). When a chlorophyll pair, or dimer, absorbs light energy, it has more energy than it originally possessed and is referred to as being in an excited state. In this excited state, electrons associated with the chlorophyll dimer are not as tightly bound and can be easily removed. The chlorophyll dimer is called a donor species because it donates an electron to another molecule. The recipient of these electrons is called an acceptor and it is reduced on acceptance of the electrons released by the chlorophyll dimer. The electrons that are passed from one molecule to another are "unpaired," since they exist as single negative charges. Creation of a pair of charges, one positive and one negative, following photoexcitation is the way that plants convert light energy into chemical energy.

Donor and acceptor molecules work together to stabilize opposite charges. Figures 8 and 9 show that after light is absorbed, an initial donor, a pair of chlorphyll molecules called P680, is photoexcited. P680 then donates an electron to the first acceptor molecule, which is a chemical relation of chlorophyll called Pheo, through a process known as charge separation. Spatial separation of the positive charge on P680 from the negative charge on Pheo stabilizes the opposite charges. When close together in space, the likelihood that two opposite charges will combine to form a neutral species is high. Annihilation of the positive charge by reaction with the negative charge wastes the absorbed light energy. However, if the positive and negative charges can be quickly separated over a distance, the probability of forming a neutral species through recombination drops significantly.

One way to envision how photosynthetic organisms separate charges by a distance is to imagine that the two charged species migrate away from each other, a behavior similar to that of two charged species in a solution. Unfortunately the two charged species in a photosynthetic reaction center cannot move appreciably because both are tightly held in place by the protein to which they are bound. The remedy that photosynthetic organisms have adopted is one in which the charges are passed in rapid succession by electron transfer from one molecule to another molecule. Photoexcitation, charge separation, electron transfer and charge stabilization are crucial components of how plants convert solar energy to chemical energy.

How Do We Study Photosynthesis?

At this point, we have mentioned the way in which photosynthetic organisms harvest light and the initial electron-transfer steps that they use to convert light energy into chemical energy. What we have not discussed is how these processes are studied in the laboratory.

There are many different methods for studying photosynthesis, two of which are particularly useful for studying the chemical reactions leading to O₂ production. The first, electron-paramagnetic-resonance (EPR) spectroscopy, detects unpaired electrons by observing changes in an electron's properties when a magnetic field is applied to a sample. By itself, the electron has a small magnetic field, similar to a small bar magnet. When a large external magnetic field is applied, the electron responds by orienting itself with the external magnetic field. In a reaction center like PSII, EPR spectroscopy can be used to detect the unpaired electrons generated by charge separation and charge stabilization.

One of the most valuable aspects of EPR spectroscopy is identification of the molecules on which unpaired electrons reside. This is possible because electrons are affected by small internal magnetic fields in addition to the applied external magnetic field and because different molecules have characteristic small internal magnetic fields. For example, an unpaired electron found on a chlorophyll molecule produces a different signal by EPR spectroscopy than an unpaired electron found on a metal atom. For the purposes of experiments using PSII, EPR spectroscopy can help determine the location of an electron in the reaction center by identifying the molecule on which the electron resides. This is particularly helpful for obtaining information about the chemical intermediates in the O₂-forming reactions of PSII.

The second technique makes use of x rays. Like visible light, x rays are absorbed by the sample when it is placed in an x-ray beam. If a protein contains elements possessing atomic numbers greater than 19 (potassium), these atoms will absorb x rays of a characteristic energy. The spectrum obtained from x-ray absorption spectroscopy (XAS) can give information about the oxidation state of the absorbing atom, the types and distances of other atoms near the absorbing atom and the symmetry of the absorbing atom's environment. Further information can be obtained by comparing XAS spectra of the protein to those obtained for discrete molecular complexes.

A unique feature of O₂ production is that it is initiated by light. This means that EPR and XAS spectra can be collected in the dark before any reactions take place and then collected again after a sample has been exposed to light. Subtracting the spectrum collected in the dark from the spectrum collected in the light yields a final spectrum that contains only features associated with light-dependent reactions. In this way, investigators studying photosynthetic O₂ production can selectively monitor reactions in their samples by controlling the conditions (temperature, light exposure) of the experiment.

Photosystem II

Understanding O₂ production in plant photosynthesis involves understanding both the structure and the function of the PSII protein complex. Determination of the structure of PSII would make it possible to locate essential parts of the protein relative to each other. In effect, we would have a map of where everything is located in PSII. Unfortunately, until recently, the large amounts of highly purified PSII required for structure-determination measurements have been difficult to obtain. Luckily, this has not been as severe a limitation as one might imagine because the structure of the reaction center from bacteria has been solved, and parts of that structure are similar to PSII. Of course, the drawback of comparing bacteria and higher plants is that bacteria do not generate O₂, and, therefore, the O₂-evolving components that we are attempting to study are not present in the structure of the bacterial reaction center. Even without a complete structural model of PSII, we can still study the way in which the protein functions by using spectroscopic techniques. These techniques have revealed that PSII contains at least five distinct nonprotein compounds, called cofactors, and a tyrosine amino acid residue from the surrounding protein, which directly participate in O₂ evolution. These cofactors are small molecules or metal ions that are bound within the protein. They relay electrons to one another in a series of oxidation and reduction reactions (also called redox reactions) and catalyze reactions of PSII. In addition to these six "involved" redox centers, PSII contains multiple other cofactors whose functions under physiological conditions are still unknown.

A model of the arrangement of these various cofactors in PSII, based on the information gathered so far, has been assembled into Figures 8 and 10, which show the six redox centers that directly participate in O₂ evolution and the direction in which electrons are transferred. What emerges is a sort of relay in which electrons are transferred from one to another molecule in the PSII complex.

The relay starts with the chlorophyll dimer in PSII that absorbs light energy and initiates the charge separation. This chlorophyll dimer is called P680, so named because it is a pigment for which one of the maxima in its visible absorption spectrum occurs at 680 nanometers. A few picoseconds after photoexcitation, P680 transfers an electron to Pheo—a member of the chemical family of pheophytins and a relation to chlorophyll—to create the charge-separated state P680⁺Pheo^–. Structurally, a pheophytin is a chlorophyll molecule that lacks a central magnesium ion.

After approximately 200 picoseconds, the unpaired electron on Pheo^– is transferred to the species called Q_A, a quinone molecule with a long tail that is tightly bound to the protein. It only accepts one electron and, once it has done so, forms the semiquinone radical anion Q_A^–. In 100–200 microseconds, the electron on Q_A^– is transferred to another quinone species, designated Q_B. The Q_B binding site acts as a sort of depot for protons and electrons. In PSII, free quinone binds in the Q_B site, accepts two electrons and two protons (H⁺), leaves the Q_B site and is replaced by a new quinone molecule. Because the quinone that binds in the Q_B site eventually leaves, it is the terminal electron acceptor in the chain of electron-transfer processes in PSII. The free quinone then shuttles the protons and electrons to the cytochrome b₆/f complex in the thylakoid membrane.

The two remaining redox centers that have not been discussed are the species Tyr_Z and OEC. The abbreviation "Tyr" denotes the amino acid tyrosine, one of the amino acids in the polypeptide in which the OEC is embedded; the subscript Z is used to differentiate this tyrosine from other tyrosines in PSII. During O₂ production, Tyr_Z is reversibly oxidized and reduced. Almost as soon as the strong oxidant P680⁺ is formed, Tyr_Z rapidly donates an electron to it, thus regenerating neutral P680. Although P680⁺ removes an electron from Tyr_Z, Tyr_Z also loses a proton in the same step. The result is that Tyr_Z becomes the neutral tyrosine radical Tyr_Z^? rather than a charged tyrosine cation Tyr_Z⁺. This also means that to regenerate Tyr_Z, Tyr_Z^? must regain both an electron and a proton, which in fact happens in the very next step.

Tyr_Z^? takes an electron and a proton from the manganese-containing complex called the oxygen-evolving complex, or OEC. During this step, Tyr_Z^? returns to its neutral state, and the OEC loses an electron and a proton. To replace its lost electrons and protons, the OEC strips electrons and protons away from water—the electron donor in the light reaction of plant photosynthesis. Just as quinone is the terminal electron acceptor in PSII, water is the terminal electron donor.

The OEC is the site at which water binds and is oxidized to O₂. It is known that positively charged manganese ions are bound to PSII. It has also been found that when manganese is removed from PSII, the system no longer produces O₂. These observations suggest that water molecules bind directly to manganese atoms and then are converted to O₂. Through the use of many techniques, including EPR and x-ray spectroscopies, the OEC has been modeled as a cluster of four manganese atoms.

In 1969, before much information about the chemical nature of the OEC was known, French researcher Pierre Joliot devised an experiment to determine whether the conversion of two water molecules to one O₂ molecule happened in one step or in several. His plan was to deliver light flashes to a sample of photosynthetic algae or chloroplasts and then monitor the amount of O₂ generated after each light flash. He observed that when a sample was prepared and incubated in the dark before being exposed to the light flashes, three flashes were required before O₂ was produced. He also noted that after the first cycle of three light flashes, four light flashes were required before O₂ was detected (Figure 11).

Based on Joliot's observation, Dutch scientist Bessel Kok proposed that PSII stored the oxidizing equivalent produced by each flash of light until the four oxidizing equivalents needed to form O₂ from two molecules of H₂O had been stored. Converting this idea into a model, he devised what is called the Kok or "S-state" cycle (Figure 11). Each S state is a storage state that PSII uses to accumulate oxidizing power. Although some controversy still exists, it is commonly agreed that the S states are associated with the manganese cluster in the OEC. This has been interpreted to mean that the advance from one S state to the next requires the removal of an electron from the manganese atoms in the OEC. During one full S-state cycle, two water molecules bind to the OEC and are converted to reactive species so that by the end of the cycle O₂ is produced.

Making Models of the OEC

Because we know that the OEC contains four manganese atoms, it seems reasonable to assume that small compounds containing four manganese atoms could be synthesized in the laboratory. The aim would be to build molecules that convert water to O₂ by behaving like the manganese cluster found in the protein PSII. Unfortunately, little progress toward this specific goal has been made. Although many complexes containing manganese exist, the vast majority of them neither oxidize water nor produce O₂.

The first example of a complex that functionally mimics the manganese cluster in PSII was discovered more than two decades ago by Thomas Meyer and coworkers at the University of North Carolina at Chapel Hill; however, it does not contain manganese at all. It is a ruthenium complex containing a core of two ruthenium atoms, one oxygen atom and two water molecules (Figure 12a). The way in which this ruthenium complex evolves O₂ has begun to be unraveled, but the mechanistic details of the final O₂-formation step are still unknown. What is clear is that by removing electrons from the ruthenium atoms, the entire complex changes and eventually releases O₂.

The key intermediate in this process is a ruthenium atom bonded to an oxygen atom through a double bond, a Ru=O species. The oxygen atom bound to ruthenium via a double bond is derived from one of the water molecules that was originally bound to the complex. Creation of a Ru=O species converts the unreactive oxygen atom in water to a very reactive oxygen atom capable of forming the O₂ bond.

The second complex that has been reported to generate O₂ is a manganese complex created by linking together two large molecules that each contain one manganese atom (Figure 12b). In the resulting species, the two manganese atoms are close to each other. Complexes like these have been proposed to contain Mn=O species that are similar to the Ru=O species described above.

The final manganese-containing complex shown to produce O₂ catalytically has been developed in our laboratory. The core of our complex is similar to that found for the ruthenium complex, except that the water molecules are arranged differently. Current experiments suggest that our manganese model complex also uses a Mn=O species to produce O₂.

Water-Oxidation Chemistry of PSII

How do these small complexes compare with estimates of what the manganese cluster in PSII looks like? X-ray and EPR spectroscopies have helped to identify possible structures and oxidation states of the manganese atoms in the OEC. In particular, XAS has aided in assessing which additional atoms may be near manganese. XAS spectra of the S₁ state of the Mn₄ cluster in PSII indicate that it contains interatomic distances of 2.7 and 3.3 angstroms. In small molecule model complexes (for example, Figure 12c), which consist of a Mn₂O₂ core, the distance between manganese atoms is about 2.7 angstroms. Therefore, models that assume a distance of 2.7 angstroms for atoms in the Mn₄ cluster in PSII are based on a similar core structure.

Using a value of 3.3 angstroms for the interatomic distance gives rise to several different structural models and forms the basis of considerable controversy in the field at this time. Assuming that the 3.3-angstrom distance arises from an additional manganese–manganese pair, Melvin Klein and Kenneth Sauer at the University of California at Berkeley have proposed the model of the Mn₄ cluster in PSII shown in Figure 13.

In this model, two Mn₂O₂ cores are linked, separated by a distance of 3.3 angstroms on one side of the cluster. To attain the 3.3-angstrom distance, the Mn₂O₂ cores are connected by only one oxygen atom. The other atoms bridging the Mn₂O₂ cores are carboxylate groups derived from amino acids in the protein. Although some of the essential features of the Mn₄ cluster of PSII are probably well represented by the model in Figure 13, it should be considered a "working" model to be adapted to accommodate new ideas and results.

Models and Mechanisms

The structural model of the Mn₄ cluster of the OEC shown in Figure 13 has initiated consideration of the discrete steps involved in water oxidation and O₂ evolution. Most mechanistic proposals concentrate on the Mn₄ cluster because S-state advance correlates with O₂ production. However, a recent suggestion is that the tyrosine residue Tyr_Z, which becomes oxidized and reduced, may also be intimately involved in water oxidation. Initial measurements of the distance between Tyr_Z and the Mn₄ cluster indicated that the two species were 10–20 angstroms apart. Consequently, it was believed that Tyr_Z^? acquired just an electron from the Mn₄ cluster: The proton required for Tyr_Z^? reduction presumably originated from some other source. Now, studies conducted in our lab and others have demonstrated that Tyr_Z and the Mn₄ cluster are in close proximity. Simulations of an EPR spectrum containing features from both Tyr_Z^? and the Mn₄ cluster indicate that the distance between the two cofactors is approximately 8 angstroms.

This means that Tyr_Z is close enough to the Mn₄ cluster to participate directly in water-oxidation chemistry. Figure 13 illustrates a possible sequence of reactions that takes place between the S₃ and S₀ states during which O₂ is released. Starting with the Mn₄ cluster in the S₃ state, removal of one electron and one proton (H⁺) from Tyr_Z forms the tyrosine radical, Tyr_Z^?. The advance to the S₄ state happens when Tyr_Z^? removes an electron from the Mn₄ cluster and a proton from a hydroxide (OH^–) group bound to the Mn₄ cluster. Once an electron and a proton have been removed from the Mn₄ cluster by Tyr_Z^?, a Mn=O species can form in the S₄ state. Creation of a Mn=O species in the Mn₄ cluster in PSII is based on analogy to the chemistry exhibited by small model complexes that generate Mn=O species.

In the O₂ bond-forming step, a hydroxide (OH^–) group attacks the Mn=O species. This hydroxide group is not expected to be diffusing freely in solution. Instead, it may be bound to a calcium ion before it is delivered to the Mn=O group. This may explain why calcium is required for maximal O₂ production. The end result is collapse of the Mn₄ cluster back to the S₀ state and the concomitant release of O₂.

Many factors have contributed to our increased understanding of photosynthetic O₂ evolution. Continued spectroscopic scrutiny of the Mn₄ cluster in PSII can be used to refine mechanistic proposals. Study of small-molecule model complexes that generate O₂ provides insight into the feasibility of mechanistic proposals of water oxidation and O₂ evolution in plants. The discovery of specific strains of cyanobacteria that do not require Photosystem II for growth means that genetic manipulation at discrete sites ("site-directed mutagenesis") is now used to probe the structure, location and function of many of the cofactors in PSII. And, in the not too distant future, advances in purification procedures of PSII may permit measurements that could eventually yield the entire structure of PSII. The question of how plants accomplish the incredible feat of converting water to O₂ remains a challenging and rewarding problem.

Acknowledgment

This work was supported by the National Institutes of Health (GM 32715 and GM 36442, and a pre-doctoral traineeship to Szalai, GM08283).