of specific amino acids. The reaction rate of the proton release strongly depended on the redox condi-
tions as well as on the pH in specific cases [28,29]. Manganese as an essential cofactor in photosystem
II has some specific properties that made it extraordinarily well suited for an important role in the re-
dox system of photosynthesis. The most significant one might be that manganese occurs in four differ-
ent valence states in covalent binding situations with oxygen; in complexes six and maximally seven
different valence states have been described. In fact, it has been found, e.g., by analyses of manganese
K-edge X-ray absorption near edge structure (XANES) measurements, that during the light-induced cy-
cling through the “Kok clock” valence states from the essential manganese cluster within photosystem
II appear to change systematically. (Unfortunately, the scientific results are not unequivocal and there
is still a strong debate about the details and even about whether there is really a clear correlation be-
tween a given valence state and a corresponding S state of the OEC.)
One of the essential questions in this context was whether substrate water is oxidized with or with-
out an obligatory intermediate and in which state of the cycle a specific water molecule has to be irre-
versibly bound in order to be oxidized upon a following flash. For a long time this question could not be
answered because substrate water molecules could not be discriminated depending on their binding in dif-
ferent redox states. Only mass spectrometric analyses with the application of stable isotopes containing
water molecules (H 218 O) resolved this problem (Figure 8) [30,31]. The trick was to add the H 218 O only
after preflashing the photosynthetic assay with (in the initial experiment) two preflashes. In this way the
reaction centers were transferred from the dark stable S 1 to S 3. Onto this prefabricated S 3 , substrate wa-
ter in the form of H 218 O was provided and one analyzing flash was fired. As can be seen from Figure 8,
this analyzing flash yielded a significant amount of isotopic molecular oxygen (^18 O 2 ). This result meant
that substrate water had been exchanged in the highly oxidized S 3 state! The important conclusion for the
mechanism of photosynthetic water oxidation was that there is not necessarily an oxygen precursor or par-
tially oxidized water, which had generally been assumed earlier. Molecular water can be oxidized by the
appropriate redox conditions and a singleflash [30,31].
Mass spectrometry proved important in plant physiological research because of further advantages
and specificities. With the choice of a suitable isotope distribution and composition of both liquid and gas
phases in a given reaction assay, it became possible to directly record oxygen evolution and oxygen up-
take reactions independently, concomitantly, and nearly without interference. Experiments have shown
for the first time the blue light–enhanced respiration of algae under the conditions of running photosyn-
thesis, i.e., during light-induced oxygen evolution [32]. This result was achieved by recording photosyn-
thetic oxygen evolution from H 216 O as^16 O 2 (m/e32) and the respiratory oxygen uptake from an arti-
ficially installed^18 O 2 atmosphere as^18 O 2 (m/e36).
Appropriate mass spectrometric assays have been developed and applied for measurements of dif-
ferent light-induced gas exchange reactions in algae and plants. The carbon dioxide metabolism has been
investigated, e.g., by means of the mass spectrometric setup of Badger [33]. In our laboratory, nitrogen
fixation by blue-green algae (cyanobacteria) could be directly followed and quantified as^15 N 2 uptake at
m/e30 depending on the presence (or rather the absence) of a combined nitrogen source in the medium.
Trivially, light-induced nitrogen fixation was observed only with cultures grown without an N source
[34].
The oxygen partial pressure of the atmosphere (natural or artificial) seems to be much more relevant
for optimal functioning of photosynthesis than was originally expected. Normally, the oxygen concen-
tration was considered essential “only” for the reaction rates of respiratory processes (see later). It ap-
peared, however, that oxygenic photosynthesis did not function in the complete absence of molecular
oxygen (Figure 9). Small but distinct amounts appeared necessary to catalyze a normal water oxidation
reaction [2]. It was shown in our laboratory that about four molecules of O 2 are required per reaction cen-
ter and that oxygen is bound in a cooperative manner (Figure 10). Our observation immediately calls to
mind the binding properties of hemoglobin in human physiology and zoophysiology. (It was mentioned
in the introduction that hemoglobin even appears to play a direct role in plant physiology [18].) The re-
quirement of catalytic amounts of oxygen for the functioning of the OEC has been described in detail for
cyanobacteria, but it can be observed with cell suspensions from higher plants and also with green algae.
Thus, it might be taken as a general feature of oxygenic photosynthesis. One of the recent ideas about how
oxygen, molecular water, and hydrogen peroxide might interact in the immediate vicinity of the oxygen-
evolving complex has been extensively discussed [2].
PHOTOSYNTHETIC GAS EXCHANGE AND RESPIRATION 307