Molecular Mechanisms of Photosynthesis. Robert E. Blankenship
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chemical process. This principle was first clearly set forth in the 1930s by the Dutch microbiologist Cornelis van Niel (1897–1985), working at Stanford University. Van Niel carried out a series of experiments on the metabolic characteristics of non‐oxygen‐evolving (anoxygenic) photosynthetic bacteria (van Niel, 1941). These organisms contain bacteriochlorophylls, pigments related to, but distinct from, the chlorophylls contained in cyanobacteria, algae, and plants. They assimilate CO2 into organic matter, but do not produce molecular oxygen. In order for these bacteria to assimilate CO2, they must be supplied with a reducing compound. Many different compounds will suffice, most notably H2S, which is first oxidized to elemental sulfur and then further oxidized. In place of H2S, a variety of organic compounds can also be utilized, or even molecular hydrogen. Van Niel's seminal contribution was the recognition that these compounds could all be represented by the general formula H2A, and that the overall equation of photosynthesis could be reformulated in a more general way as follows:
(3.4)
The oxygen‐evolving form of photosynthesis can then be seen as a special case of this more general formulation, in which H2O is H2A and O2 is 2A. When presented in this manner, the redox nature of photosynthesis is much more obvious. In fact, it is a simple further step to separate the oxidation and reduction into two chemical equations, one for the oxidation and the other for the reduction:
This separation into oxidation and reduction reactions leads to a number of important predictions. First, it suggests that the two processes might possibly be physically or temporally separated. Indeed, this was found to be the case by Hill in his classic experiments on the use of artificial electron acceptors, which will be discussed in the next paragraph. A second prediction is that the oxygen produced by oxygen‐evolving photosynthesis comes from H2O and not from CO2. This is indeed the case, although it took many years for this fact to be established unequivocally, because of the rapid exchange of oxygen between CO2 and H2O via carbonic acid, H2CO3, which is constantly forming and breaking down. Van Niel correctly imagined that the oxidation and reduction reactions of Eqs. (3.5a) and (3.5b) were not the primary processes carried out by light, but were, rather, the result of a primary oxidant and reductant generated in the light, which went on to react with the substrates to form the products. However, some of van Niel's detailed ideas about the nature of the primary oxidant and reductant were incorrect, and he was overly rigid in insisting that all assimilated carbon had first to be oxidized all the way to CO2 before it could be subsequently reduced. Nevertheless, the truth of the basic principle of photosynthesis as a light‐induced redox process was unequivocally established by van Niel's work, along with that of other scientists of the time, notably Hans Gaffron (1902–1979).
3.7.2 The Hill reaction: separation of oxidation and reduction reactions
In the 1930s, Robert (Robin) Hill (1899–1991), working at Cambridge University, was able to separate and investigate individually the oxidation and reduction reactions of photosynthesis. This taking apart of a complex system into individual parts, and investigation of them in detail in the absence of the complexities of a living cell is a cornerstone of biochemistry, often called reductionism. Hill established that it was possible to restore high rates of oxygen evolution to chloroplast suspensions if the latter were supplied with any of a number of artificial electron acceptors (Hill, 1939). Initially, he used ferric salts, and the Fe3+ was reduced to Fe2+ through the action of light, at the same time producing O2. The reduction in artificial acceptors with concomitant O2 production is today known as the Hill reaction. An example of the Hill reaction is given in Eq. (3.6):
Hill measured the O2 in an ingenious way, which is worth relating if only to give an idea of the remarkable advances made by many of the pioneers of the field despite their primitive instrumentation. Hill obtained whole blood from a slaughterhouse, which has a dark blue color when deoxygenated and bright red color when oxygenated. He combined this with his chloroplast preparation and illuminated the mixture, monitoring the degree of oxygenation of the blood using a hand‐held spectroscope. At first, the results were disappointing, because the sample produced little oxygen. It is now clear that this was because the outer chloroplast envelope membranes were broken during the preparation, and the enzymes needed for CO2 assimilation were lost. In searching for the factors needed to restore the lost activity, Hill made a fundamental discovery: namely, that it was possible to replace the reduction of CO2 with the reduction of artificial electron acceptors, thereby restoring high rates of O2 production. The physiological compound that acts as the light‐driven electron acceptor facilitating CO2 production is NADP+, the oxidized form of nicotinamide adenine dinucleotide phosphate. The reduced form of this compound, NADPH, then serves as the reductant for CO2 assimilation.
Hill did not set out to discover the reaction that bears his name. Instead, he was trying to establish whether an isolated chloroplast was capable of the complete process of photosynthesis, which was an important issue at the time. In fact, it is quite difficult to isolate chloroplasts with the envelope membranes still intact, and this was not routinely achieved until the mid‐1960s. This is a good example of Louis Pasteur's famous saying that “fortune favors the prepared mind.”
3.8 The Emerson and Arnold experiments
In 1932, Robert Emerson (1903–1959) and his undergraduate research student at the California Institute of Technology, William Arnold (1904–2001), published two seminal papers. They were the first scientists to exploit the use of very short flashes of light to probe photosynthesis. At the time, this was extremely difficult technically; one did not just order a flash instrument from a scientific supply house. Emerson and Arnold built their own apparatus from automobile ignition points, capacitors, neon lights, and even a hot plate (which served as a ballast resistor) (Myers, 1994). Using this device, they were able to obtain flash durations as short as 10 μs. Emerson and Arnold utilized the green alga Chlorella pyrenoidosa as their experimental organism and measured oxygen evolution using manometers, U‐shaped tubes containing liquids in which the level was used to determine the volume of gas produced or consumed. Emerson was a master in the techniques of manometry, which he had learned from his former mentor, Otto Warburg, and could measure minute changes of O2 with great accuracy.
In the first series of experiments (Emerson and Arnold, 1932a), they varied the time between flashes and found that if there was a long time between flashes, then the yield of O2 per flash was independent of the time between flashes and did not depend on temperature between 1 and 25 °C (Fig. 3.2). With shorter times between flashes, the yield fell off dramatically at the lower temperature, but did not change at the higher temperature. This result was elegantly interpreted as evidence that photosynthesis involved both a light stage and a dark stage. The light stage, which we now refer to as a photochemical reaction, can happen extremely rapidly and is not dependent on temperature. The dark stage, which we now know to be a series of enzymatic reactions, is slower and,