18O isotope exchange measurements of photosystem II (PSII) in thylakoids from wild-type and mutant Synechocystis have been performed to investigate binding of substrate water to the high-affinity Mn4 site in the oxygen-evolving complex (OEC). The mutants investigated were D1-D170H, a mutation of a direct ligand to the Mn4 ion, and D1-D61N, a mutation in the second coordination sphere. The substrate water 18O exchange rates for D61N were found to be 0.16±0.02s-1 and 3.03±0.32s-1 for the slow and fast phases of exchange, respectively, compared with 0.47±0.04s-1 and 19.7±1.3s-1 for the wild-type. The D1-D170H rates were found to be 0.70±0.16s-1 and 24.4±4.6s-1 and thus are almost within the error limits for the wild-type rates. The results from the D1-D170H mutant indicate that the high-affinity Mn4 site does not directly bind to the substrate water molecule in slow exchange, but the binding of non-substrate water to this Mn ion cannot be excluded. The results from the D61N mutation show an interaction with both substrate water molecules, which could be an indication that D61 is involved in a hydrogen bonding network with the substrate water. Our results provide limitations as to where the two substrate water molecules bind in the OEC of PSII.
|Journal||Philosophical Transaction of the Royal Society: B- Biological Sciences|
|Publication status||Published - 2008|