Molecular Mechanisms of Photosynthesis. Robert E. Blankenship
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depends on temperature activation to proceed. These results, which were partially anticipated by other scientists using continuous and crude intermittent light methods, were readily interpretable on the basis of the understanding of photosynthesis available at the time. However, they did no more than set the stage for the remarkable result that Emerson and Arnold found in their second set of experiments, in which they examined the light‐intensity dependence of the photochemical reaction.
Figure 3.2 Emerson and Arnold's experiment establishing a light stage and a dark stage of photosynthesis. Time refers to the time between flashes.
Source: Emerson and Arnold (1932a)/Rockefeller University Press.
For the second series of experiments, Emerson and Arnold (1932b) varied the light intensity of the flashes, using a flash spacing that they knew from the first series of experiments was long enough for the dark enzymatic reactions to proceed to completion. This experimental protocol enabled them to isolate the photochemical reaction and study it without interference from the later steps. At very low intensities the yield of O2 per flash was low and depended linearly on the flash energy. However, at higher intensities, the curve saturated, so additional flash energy gave no additional O2 (Fig. 3.3).
Most experimenters would have been satisfied with just establishing that the curve saturated at a high light intensity. After all, if at very high light intensity every chlorophyll molecule absorbs a photon and produces photoproducts, then one expects that additional light will give no further products until the slow enzymatic reaction restores the chlorophyll to an active state. The beauty of the experiment lies in the fact that Emerson and Arnold took great pains to obtain a quantitative measure of how much O2 was produced per chlorophyll in the sample. This may sound like a simple matter, but at the time the quantitative absorption properties of chlorophyll were not well known, so Emerson and Arnold had to determine this in order to know how many chlorophyll molecules were in their sample. The measurement of the amount of O2 produced was easier, utilizing the resulting volume and the known properties of gases.
Figure 3.3 Emerson and Arnold's experiment establishing the light saturation curve for photosynthesis in flashing light.
Source: Emerson and Arnold (1932b) (p. 1940)/Rockefeller University Press.
The final result was a huge surprise. Only one O2 was produced for every 2500 chlorophyll molecules, far less than the one per chlorophyll that was expected! Their findings were difficult to reconcile with the common idea at the time that each chlorophyll molecule directly reduced CO2. It was many years before the true significance of this experiment was appreciated. We now know that the vast majority of chlorophyll molecules act as antennas and function only to collect light, transferring the energy to a special chlorophyll molecule (which is part of a protein complex known as the reaction center) that actually does the photochemistry. This is like a satellite dish, which collects radio waves and sends them to a receiver, where the signal is detected (Fig. 1.3). However, in 1932, Emerson and Arnold were only able to propose that a large number of chlorophyll molecules acted as a group to carry out photosynthesis, although it was not clear how this cooperation came about. The collection of chlorophyll molecules and associated enzymes came to be known as a photosynthetic unit, a term proposed by Gaffron and Wohl (1936). We will explore the details of the structure and function of antennas, reaction centers, and other components of the photosynthetic unit in later chapters.
3.9 The controversy over the quantum requirement of photosynthesis
In the 1940s and 1950s, a controversy raged in the field of photosynthesis over the minimum quantum requirement for the process (Nickelsen and Govindjee, 2011). The quantum requirement is the number of photons that need to be absorbed for a photochemical process to take place. It is the reciprocal of the quantum yield. Otto Warburg, the Nobel prize‐winning German biochemist who had developed the manometric techniques that were standard for measurement in these experiments, steadfastly maintained that the minimal quantum requirement for photosynthesis was three to four photons per O2 evolved. Essentially everyone else obtained much higher values, in the range of 8–10 photons per O2 produced. Foremost among these researchers was Warburg's former student, Emerson, who had earlier carried out the experiments with Arnold described above. The argument raged on for many years and was really only settled after Emerson's premature death in an airplane crash in 1959, followed by Warburg's death in 1970.
This disagreement may seem to be only an academic issue, but the outcome was essential to the development of a deeper understanding of the underlying chemical mechanism of photosynthesis. The discussion really boils down to energetics. The energy content of the three photons that Warburg thought were all that was needed is just barely enough to account for the free energy difference between the reactants and the products (see Chapter 13). Warburg was pleased with this result, which coincided with his nineteenth‐century romantic view of nature, summarized by the comment often attributed to him: “In a perfect world photosynthesis must be perfect.” Emerson's view was more practical, and thousands of subsequent measurements in many laboratories have supported his higher numbers for the quantum requirement for photosynthesis.
Exactly why Warburg obtained the results he did is still not entirely clear, but it is thought to have to do with interactions of photosynthesis and respiration, including transient “gushes” and “gulps.” The measurement shows only net oxygen production; to get the rate of photosynthesis, it is necessary to correct for the rate of respiration. If the rate of respiration is unchanged between light and dark, this correction will be accurate; but if photosynthesis inhibits respiration (as some modern evidence suggests), the correction will lead to erroneously low values for the quantum requirement. In retrospect, it is clear that Warburg, despite being a brilliant experimenter and very experienced professional scientist of the highest rank, fell into the very human trap of thinking that he knew what the answer should be and then not being sufficiently objective in evaluating his own experiments.
Unfortunately, the quantum requirement controversy took up the enormous time and effort of many of the foremost scientists of the day and didn't directly lead to a new understanding of the mechanism of photosynthesis. However, in the process of thoroughly examining the conditions required for the measurement of the quantum requirement for photosynthesis, some important new discoveries were made, which ultimately did lead to a much deeper understanding. Chief among these were the phenomena known as the “red drop” and “enhancement.”
3.10 The red drop and the Emerson enhancement effect
As part of his attempt to settle the controversy with Warburg, Emerson and coworkers made careful measurements in the 1940s of the quantum requirements for photosynthesis as a function of wavelength and obtained a most remarkable result. As the wavelength of light utilized for the experiment approached the red edge of the absorption of the chlorophyll, the quantum requirement went up dramatically. The action spectrum for photosynthesis is remarkably congruent with the absorption spectrum throughout much of the visible wavelength range, but drops off more quickly in this far‐red region (Fig. 3.4). An action spectrum is a plot of the effectiveness of light to cause a given effect, in this case oxygen evolution, versus the wavelength