Photosynthetic water oxidation by photosystem II is mediated by a Mn4 cluster, a cofactor X still chemically ill-defined, and a tyrosine, YZ (D1-Tyr161). Before the final reaction with water proceeds to yield O2 (transition S4-->S0), two oxidizing equivalents are stored on Mn4 (S0-->S1-->S2), a third on X (S2-->S3), and a forth on YZ(S3-->S4). It has been proposed that YZ functions as a pure electron transmitter between Mn4X and P680, or, more recently, that it acts as an abstractor of hydrogen from bound water. We scrutinized the coupling of electron and proton transfer during the oxidation of YZ in PSII core particles with intact or impaired oxygen-evolving capacity. The rates of electron transfer to P680+, of electrochromism, and of pH transients were determined as a function of the pH, the temperature, and the H/D ratio. In oxygen-evolving material, we found only evidence for electrostatically induced proton release from peripheral amino acid residues but not from YZox itself. The positive charge stayed near YZox, and the rate of electron transfer was nearly independent of the pH. In core particles with an impaired Mn4 cluster, on the other hand, the rate of the electron transfer became strictly dependent on the protonation state of a single base (pK approximately 7). At pH < 7, the rate of electron transfer revealed the same slow rate (t1/2 approximately 35 microseconds) as that of proton release into the bulk. The deposition of a positive charge around YZox was no longer detected. A large H/D isotope effect (approximately 2.5) on these rates was also indicative of a steering of electron abstraction by proton transfer. That YZox was deprotonated into the bulk in inactive but not in oxygen-evolving material argues against the proposed role of YZox as an acceptor of hydrogen from water. Instead, the positive charge in its vicinity may shift the equilibrium from bound water to bound peroxide upon S3-->S4 as a prerequisite for the formation of oxygen upon S4-->S0.

Photosystem II (PSII) oxidizes two water molecules to yield dioxygen plus four protons. Dioxygen is released during the last out of four sequential oxidation steps of the catalytic centre (S 0 ⇒ S 1 , S 1 ⇒ S 2 , S 2 ⇒ S 3 , S 3 ⇒ S 4 → S 0 ). The release of the chemically produced protons is blurred by transient, highly variable and electrostatically triggered proton transfer at the periphery (Bohr effect). The extent of the latter transiently amounts to more than one H ϩ /e Ϫ under certain conditions and this is understood in terms of electrostatics. By kinetic analyses of electron-proton transfer and electrochromism, we discriminated between Bohr-effect and chemically produced protons and arrived at a distribution of the latter over the oxidation steps of 1 : 0 : 1 : 2. During the oxidation of tyr-161 on subunit D1 (Y Z ), its phenolic proton is not normally released into the bulk. Instead, it is shared with and confined in a hydrogenbonded cluster. This notion is difficult to reconcile with proposed mechanisms where Y Z acts as a hydrogen acceptor for bound water. Only in manganese (Mn) depleted PSII is the proton released into the bulk and this changes the rate of electron transfer between Y Z and the primary donor of PSII P ϩ 680 from electron to proton controlled. D1-His190, the proposed centre of the hydrogen-bonded cluster around Y Z , is probably further remote from Y Z than previously thought, because substitution of D1-Glu189, its direct neighbour, by Gln, Arg or Lys is without effect on the electron transfer from Y Z to P

No abstract

The catalytic Mn cluster of the photosynthetic oxygen-evolving system is oxidized via a tyrosine, Y(Z), by a photooxidized chlorophyll a moiety, P(+)(680). The rapid reduction of P(+)(680) by Y(Z) in nanoseconds requires the intactness of an acid/base cluster around Y(Z) with an apparent functional pK of <5. The removal of Mn (together with bound Ca) shifts the pK of the acid/base cluster from the acid into the neutral pH range. At alkaline pH the electron transfer (ET) from Y(Z) to P(+)(680) is still rapid (<1 micros), whereas at acid pH the ET is much slower (10-100 micros) and steered by proton release. In the intermediate pH domain one observes a mix of these kinetic components (see R. Ahlbrink, M. Haumann, D. Cherepanov, O. Bögershausen, A. Mulkidjanian, W. Junge, Biochemistry 37 (1998)). The overall kinetics of P(680)(+) reduction by Y(Z) in Mn-depleted photosystem II (PS II) has been previously shown to be slowed down by divalent cations (added at >10 microM), namely: Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+) (C.W. Hoganson, P.A. Casey, O. Hansson, Biochim. Biophys. Acta 1057 (1991)). Using Mn-depleted PS II core particles from pea as starting material, we re-investigated this phenomenon at nanosecond resolution, aiming at the effect of divalent cations on the particular kinetic components of P(+)(680) reduction. To our surprise we found only the slower, proton steered component retarded by some added cations (namely Co(2+)/Zn(2+)>Fe(2+)>Mn(2+)). Neither the fast component nor the apparent pK of the acid/base cluster around Y(Z) was affected. Apparently, the divalent cations acted (electrostatically) on the proton release channel that connects the oxygen-evolving complex with the bulk water, but not on the ET between Y(Z) and P(+)(680), proper. Contrastingly, Ca(2+) and Mg(2+), when added at >5 mM, accelerated the slow component of P(+)(680) reduction by Y(Z) and shifted the apparent pK of Y(Z) from 7.4 to 6.6 and 6.7, respectively. It was evident that the binding site(s) for added Ca(2+) and Mg(2+) were close to Y(Z) proper. The data obtained are discussed in relation to the nature of the metal-binding sites in photosystem II.

No abstract