FEATURE ARTICLE
How Plants Produce Dioxygen
At its core, oxygen production comes down to the chemistry of a poorly understood manganese-containing complex in the membranes of plant chloroplasts
Veronika Szalai, Gary Brudvig
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.
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