The Two Substrate−Water Molecules Are Already Bound to the Oxygen-Evolving Complex in the S2 State of Photosystem II

• Published in: Biochemistry (Easton) 2002-11, Vol.41 (44), p.13328-13334
• Main Authors: Hendry, Garth, Wydrzynski, Tom
• Format: Article
• Language: English
• Subjects: Binding Sites; Calcium - chemistry; Deuterium Oxide - chemistry; Electrophoresis, Polyacrylamide Gel; Kinetics; Oxygen - chemistry; Oxygen Isotopes - chemistry; Photolysis; Photosynthetic Reaction Center Complex Proteins - chemistry; Photosystem II Protein Complex; Plant Proteins; Spinacia oleracea; Water - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi026246t

The first direct evidence which shows that both substrate−water molecules are bound to the O2-evolving catalytic site in the S2 state of photosystem II (PSII) is presented. Rapid 18O isotope exchange measurements between H2 18O incubated in the S2 state of PSII-enriched membrane samples and the photogenerated O2 reveal a fast and a slow phase of exchange at m/e 34 (which measures the level of the 16O18O product). The rate constant for the slow phase of exchange (34 k 1) equals 1.9 ± 0.3 s-1 at 10 °C, while the fast phase of exchange is unresolved by our current experimental setup (34 k 2 ≥ 175 s-1). The unresolvable fast phase has left open the possibility that the second substrate−water molecule binds to the catalytic site only after the formation of the S3 state [Hillier, W., and Wydrzynski, T. (2000) Biochemistry 39, 4399−4405]. However, for PSII samples depleted of the 17 and 23 kDa extrinsic proteins (Ex-depleted PSII), two completely resolvable phases of 18O exchange are observed in the S2 state of the residual activity, with the following rate constants:  34 k 1 = 2.6 ± 0.3 s-1 and 34 k 2 = 120 ± 14 s-1 at 10 °C. Upon addition of 15 mM CaCl2 to Ex-depleted PSII, the O2 evolution activity increases to ∼80% of the control level, while the two resolvable phases of exchange remain the same. In measurements of Ex-depleted PSII at m/e 36 (which measures the level of the 18O18O product), only a single phase of exchange is observed in the S2 state, with a rate constant (36 k 1 = 2.5 ± 0.2 s-1) that is identical to the slow rate of exchange in the m/e 34 data. Taken together, these results show that the fast phase of 18O exchange is specifically slowed by the removal of the 17 and 23 kDa extrinsic proteins and that the two substrate−water molecules must be bound to independent sites already in the S2 state. In contrast, the 18O exchange behavior in the S1 state of Ex-depleted PSII is no different from what is observed for the control, with or without the addition of CaCl2. Since the fast phase of exchange in the S1 state is unresolved (i.e., 34 k 2 > 100 s-1), the possibility remains that the second substrate−water molecule binds to the catalytic site only after the formation of the S2 state. The role of the 17 and 23 kDa extrinsic proteins in establishing an asymmetric dielectric environment around the substrate binding sites is discussed.