The rates of proton uptake and electron transfer at the reducing side of photosystem II in thylakoids

AbstractProton and electron transfer at the reducing side of photosystem II of green plants was studied under flashing light, the former at improved time resolution by using Neutral red. The rates of electron transfer within QAFeQB were determined by pump-probe flashes through electrochromic transients. The extent of proton binding was about 1 H+/e−. The rates of proton transfer were proportional to the concentration of Neutral red (collisional transfer), whereas the rates of electron transfer out of Q−A and from QAFeQ−B to the cytochrome b6f complex were constant. The half-rise times of electron transfer (τe) and the apparent times of proton binding (τh) at 30 μM Neutral red were: Q−A ⇒ FeIIIQB (τe ⩽ 100 μs, τ, 230 μs); Q−A ⇒ FeIIQB (τe = 150 μs, τh = 760 μs); and Q−A ⇒ FeIIQ−B (τe = 620 μs, τh = 310 μs)

Evidence for impaired hydrogen-bonding of tyrosine YZ in calcium-depleted Photosystem II

AbstractPhotosystem II (PS II) evolves oxygen from two bound water molecules in a four-stepped reaction that is driven by four quanta of light, each oxidizing the chlorophyll moiety P680 to yield P+680. When starting from its dark equilibrium (mainly state S1), the catalytic center can be clocked through its redox states (S0…S4) by a series of short flashes of light. The center involves at least a Mn4-cluster and a special tyrosine residue, named YZ, as redox cofactors plus two essential ionic cofactors, Cl− and Ca2+. Centers which have lost Ca2+ do not evolve oxygen. We investigated the stepped progression in dark-adapted PS II core particles after the removal of Ca2+. YZ was oxidized from the first flash on. The difference spectrum of YZ→YoxZ differed from the one in competent centers, where it has been ascribed to a hydrogen-bonded tyrosinate. The rate of the electron transfer from YZ to P+680 was slowed down by three orders of magnitude and its kinetic isotope effect rose up from 1.1 to 2.5. Proton release into the bulk was now a prerequisite for the electron transfer from YZ to P+680. On the basis of these results and similar effects in Mn-(plus Ca2+-)depleted PS II (M. Haumann et al., Biochemistry, 38 (1999) 1258–1267) we conclude that the presence of Ca2+ in the catalytic center is required to tune the apparent pK of a base cluster, B, to which YZ is linked by hydrogen bonds. The deposition of a proton on B within close proximity of YZ (not its release into the bulk!) is a necessary condition for the reduction in nanoseconds of P+680 and for the functioning of water oxidation. The removal of Ca2+ rises the pK of B, thereby disturbing the hydrogen bonded structure of YZB

Rapid proton transfer under flashing light at both functional sides of dark-adapted Photosystem II particles

AbstractBy exposing dark-adapted Photosystem II core particles to a series of light flashes, we aimed at kinetic resolution of proton release during the four steps of water oxidation. The signal-to-noise ratio was improved by averaging under repetitive dark adaptation. The previously observed kinetic damping of pH-transients by particle aggregation was prevented by detergent. The complicating superimposition of protolytic events at the donor side (water oxidation) and at the acceptor side (quinone oxido-reduction) was unravelled by characterizing the rate constants of electron and proton transfer at the acceptor side (QA− · nH+ + DCBQ → QA + DCBQ− + nH+: k = 1.7 · 106 M−1 //2 DCBQ− + 2H+ → DCBQ + DCBQH2: k = 4 · 108 M−1 s−1). Contrasting with the pronounced period of four oscillations of the oxygen-evolving centre, the extent of proton release was practically constant. The apparent half-rise time of the stepped acidification was shortened upon lowering of the pH (250 μs at pH 7.5, 70 μs at pH 6.0 and 12 μs at pH 5.2). This kinetic behaviour was independent of the nature and the concentration of the added pH-indicator. We conclude that this reflects the protolysis of several electrostatically interacting acids at the surface of the protein in response to a new positive charge on YZ+, and persisting upon electron transfer from the manganese cluster to YZ+

Chloride-depletion of photosynthetic water oxidase No proton release during the second oxidation step, S2∗ ⇒ S3∗, and a transmembrane radical pair recombination from the third on

AbstractChloride depletion blocks the normal four-step progress of photosynthetic water oxidation. We studied proton release in chloride-depleted thylakoids which were dark-adapted and excited by flashing light. Proton release was blocked from the second flash on, possibly leaving an uncompensated positive charge in the catalytic centre. The reduction of P+680 by Tyrz was still very rapid (⪡ 10 μs). From the third flash on, P+680 was reduced more slowly (70 μs200 μs), and by an electrogenic back-reaction. The uncompensated positive charge may be the reason why the rapid reduction of P+680 by Tyrz is prevented and the transmembrane charge-pair recombination is facilitated

Regulation of the F0F1-ATP synthase: The conformation of subunit ε might be determined by directionality of subunit γ rotation

AbstractF0F1-ATP synthase couples ATP synthesis/hydrolysis with transmembrane proton transport. The catalytic mechanism involves rotation of the γεc∼10-subunits complex relative to the rest of the enzyme.In the absence of protonmotive force the enzyme is inactivated by the tight binding of MgADP. Subunit ε also modulates the activity: its conformation can change from a contracted to extended form with C-terminus stretched towards F1. The latter form ihnibits ATP hydrolysis (but not synthesis).We propose that the directionality of the coiled-coil subunit γ rotation determines whether subunit ε is in contracted or extended form. Block of rotation by MgADP presumably induces the extended conformation of subunit ε. This conformation might serve as a safety lock, stabilizing the ADP-inhibited state upon de-energization and preventing spontaneous re-activation and wasteful ATP hydrolysis. The hypothesis merges the known regulatory effects of ADP, protonmotive force and conformational changes of subunit ε into a consistent picture

Photosynthetic oxygen evolution: Net charge transients as inferred from electrochromic bandshifts are independent of proton release into the medium

AbstractThe manganese containing center of the oxygen evolving complex accumulates four oxidizing equivalents in the four stepped water oxidation cycle. Based on experiments on electrochromic absorption transients and the reduction rate of the primary electron donor, P680, 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 electrochromism 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 electrochromically responsive pigments rather than responding to proton release from the periphery

Time-resolved oxygen production by PSII: chasing chemical intermediates

AbstractPhotosystem II (PSII) produces dioxygen from water in a four-stepped process, which is driven by four quanta of light and catalysed by a Mn-cluster and tyrosine Z. Oxygen is liberated during one step, coined S3⇒S0. Chemical intermediates on the way from reversibly bound water to dioxygen have not yet been tracked, however, a break in the Arrhenius plot of the oxygen-evolving step has been taken as evidence for its existence.We scrutinised the temperature dependence of (i) UV-absorption transients attributable to the reduction of the Mn-cluster and tyrosine Z by water, and (ii) polarographic transients attributable to the release of dioxygen. Using a centrifugatable and kinetically competent Pt-electrode, we observed no deviation from a linear Arrhenius plot of oxygen release in the temperature range from −2 to 32 °C, and hence no evidence, by this approach, for a sufficiently long-lived chemical intermediate. The half-rise times of oxygen release differed between Synechocystis WT* (at 20 °C: 1.35 ms) and a point mutant (D1–D61N: 13.1 ms), and the activation energies differed between species (Spinacia oleracea, 30 kJ/mol versus Synechocystis, 41 kJ/mol) and preparations (PSII membranes, 41 kJ/mol versus core complexes, 33 kJ/mol, Synechocystis).Correction for polarographic artefacts revealed, for the first time, a temperature-dependent lag-phase of the polarographic transient (duration at 20 °C: 0.45 ms, activation energy: 31 kJ/mol), which was indicative of a short-lived intermediate. It was, however, not apparent in the UV-transients. Thus the “intermediate” was probably newly formed and transiently bound oxygen

Acute reduction of uterine blood flow and fetal heart rate changes in pregnant sheep near term

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