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.
During the four-stepped catalytic cycle of water oxidation by photosystem II (PSII) molecular oxygen is released in only one of the four reaction steps whereas the release of four protons is distributed over all steps. In principle, the pattern of proton production could be taken as indicative of the partial reactions with bound water. In thylakoids the extent and rate of proton release varies as function of the redox transition and of the pH without concomitant variations of the redox pattern. The variation has allowed to discriminate between deprotonation events of peripheral amino acids (Bohr effects) as opposed to the chemical deprotonation of a particular redox cofactor, and of water. In contrast, in thylakoids grown under intermittent light, as well as in PSII core particles the pattern of proton release is flat and independent of the pH. This has been attributed to the lack in these materials of the chlorophyll a,d-binding (CAB) proteins. We now found that a thylakoid-like, oscillatory pattern of proton release was restored simply by the addition of glycerol which modifies the protein-protein interaction. Being a further proof for the electrostatic origin of the greater portion of proton release, this effect will serve as an important tool in further studies of water oxidation.
The manganese containing center of the oxygen evolving complex accumulates four oxidizing equivalents in the four stepped water oxidation cycle. Based on experiments on electrcchromic absorption transients and the reduction rate of the primary electron donor, Peso, it has been speculated that the oscillations of these variables reflect the net charge of the center as calculated from the difference. between electron abstraction and proton release into the medium. We compared proton release with electrcchromism in thylakoids and core particles, and under variation of the rate of proton release. We found no equivalent of the variations of the extents and the rates of proton release in electrochromism. The oscillatory pattern of the latter reflects the topological properties of the stepped charge storage relative to the position and orientation of electrochromicaIIy responsive pigments rather than responding to proton release from the periphery.
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