
%0 Journal Article
%A Hendry, Garth 
%A Wydrzynski, Tom 
%T Evidence for the binding of two substrate water molecules in the S2 state of photosystem II.
%J Science Access
%D 2001/01/01
%V 3
%N 1
%P -
%K 
%R https://doi.org/10.1071/SA0403271
%U https://www.publish.csiro.au/paper/SA0403271 
%X We present the first evidence to show that both substrate water molecules are bound to the catalytic site in the S2 state of photosystem II (PSII). Earlier 18O isotope exchange measurements between added H218O and the photogenerated O2 products (W. Hillier &amp; T. Wydrzynski, 2000, Biochemistry 39, 4399-4405) revealed that at least one substrate water molecule is bound in the S2 state. However, due to experimental limitations, the results left open the possibility that the second substrate water molecule only enters the catalytic site after the S2 to S3 state transition. Here we show for PSII samples depleted of the 16 and 23 kDa extrinsic proteins that both substrate water molecules are bound in the S2 state. Rapid 18O exchange measurements of the protein-depleted samples at m/e = 34 (which measures the mixed labelled 16O18O product) reveal two resolvable exchange phases in the S2 state, representing the independent exchange processes by the two bound water molecules. The exchange rates measured were 34k1 = 117 s-1 and 34k2 = 2.9 s-1 at 10 oC. The addition of 15 mM CaCl2 or MgCl2, resulted in a slowing in both phases of exchange, with rates of 34k1 = 90 s-1 and 34k2 = 1.8 s-1 or 34k1 = 74 s-1 and 34k2 = 1.6 s-1, respectively. The addition of CaCl2 increased the steady state yield of O2 evolution to 90% of the untreated control whereas MgCl2 had little effect on the residual activity, which was 20% of the control. This result indicates that under these conditions, substrate water interactions at the catalytic site are influenced by the local ionic environment. Implication of these results will be discussed. 
