Abstract
The (18)O exchange rates for the substrate water bound in the S(3) state were determined in different photosystem II sample types using time-resolved mass spectrometry. The samples included thylakoid membranes, salt-washed Triton X-100-prepared membrane fragments, and purified core complexes from spinach and cyanobacteria. For each sample type, two kinetically distinct isotopic exchange rates could be resolved, indicating that the biphasic exchange behavior for the substrate water is inherent to the O(2)-evolving catalytic site in the S(3) state. However, the fast phase of exchange became somewhat slower (by a factor of approximately 2) in NaCl-washed membrane fragments and core complexes from spinach in which the 16- and 23-kDa extrinsic proteins have been removed, compared with the corresponding rate for the intact samples. For CaCl(2)-washed membrane fragments in which the 33-kDa manganese stabilizing protein (MSP) has also been removed, the fast phase of exchange slowed down even further (by a factor of approximately 3). Interestingly, the slow phase of exchange was little affected in the samples from spinach. For core complexes prepared from Synechocystis PCC 6803 and Synechococcus elongatus, the fast and slow exchange rates were variously affected. Nevertheless, within the experimental error, nearly the same exchange rates were measured for thylakoid samples made from wild type and an MSP-lacking mutant of Synechocystis PCC 6803. This result could indicate that the MSP has a slightly different function in eukaryotic organisms compared with prokaryotic organisms. In all samples, however, the differences in the exchange rates are relatively small. Such small differences are unlikely to arise from major changes in the metal-ligand structure at the catalytic site. Rather, the observed differences may reflect subtle long range effects in which the exchange reaction coordinates become slightly altered. We discuss the results in terms of solvent penetration into photosystem II and the regional dielectric around the catalytic site.
Highlights
The oxidation of water during photosynthesis is catalyzed by the membrane-bound pigment protein complex photosystem II (PSII).1 In the net reaction, four electrons, four protons, and
Slower O2 release kinetics and increased damping are observed in PSII core preparations made from spinach, which retain the manganese stabilizing protein (MSP) yet lack the 16- and 23-kDa polypeptides [24, 25]
We have examined the 18O exchange properties of the substrate water in various PSIIcontaining preparations to consider the influence of the peripheral polypeptides on substrate binding to the catalytic site for O2 evolution
Summary
There appears to be some difference in the absolute requirement of the MSP for O2 evolution between prokaryotic and eukaryotic organisms In this respect, it is important to note that the O2 release kinetics of the ⌬psbO mutant in Synechocystis PCC 6803 is slower than in the wild type, and the O2 flash pattern is heavily damped [21, 22]. Slower O2 release kinetics and increased damping are observed in PSII core preparations made from spinach, which retain the MSP yet lack the 16- and 23-kDa polypeptides [24, 25] These results appear to show that the extrinsic polypeptides function in general to optimize the O2-evolving activity. We have extended the 18O exchange measurements to PSII-enriched membrane fragments containing a varying complement of extrinsic polypeptides, PSII core samples from spinach, thylakoid samples from ⌬psbO, wild-type strains of Synechocystis PCC 6803, and PSII core samples from Synechococcus elongatus and Synechocystis PCC 6803
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