Alternating electron and proton transfer steps in photosynthetic water oxidation.

Water oxidation by cyanobacteria, algae, and plants is pivotal in oxygenic photosynthesis, the process that powers life on Earth, and is the paradigm for engineering solar fuel-production systems. Each complete reaction cycle of photosynthetic water oxidation requires the removal of four electrons and four protons from the catalytic site, a manganese-calcium complex and its protein environment in photosystem II. In time-resolved photothermal beam deflection experiments, we monitored apparent volume changes of the photosystem II protein associated with charge creation by light-induced electron transfer (contraction) and charge-compensating proton relocation (expansion). Two previously invisible proton removal steps were detected, thereby filling two gaps in the basic reaction-cycle model of photosynthetic water oxidation. In the S(2) → S(3) transition of the classical S-state cycle, an intermediate is formed by deprotonation clearly before electron transfer to the oxidant (Y Z OX). The rate-determining elementary step (τ, approximately 30 µs at 20 °C) in the long-distance proton relocation toward the protein-water interface is characterized by a high activation energy (E(a) = 0.46 ± 0.05 eV) and strong H/D kinetic isotope effect (approximately 6). The characteristics of a proton transfer step during the S(0) → S(1) transition are similar (τ, approximately 100 µs; E(a) = 0.34 ± 0.08 eV; kinetic isotope effect, approximately 3); however, the proton removal from the Mn complex proceeds after electron transfer to . By discovery of the transient formation of two further intermediate states in the reaction cycle of photosynthetic water oxidation, a temporal sequence of strictly alternating removal of electrons and protons from the catalytic site is established.

Fast structural changes (200-900ns) may prepare the photosynthetic manganese complex for oxidation by the adjacent tyrosine radical.

The Mn complex of photosystem II (PSII) cycles through 4 semi-stable states (S(0) to S(3)). Laser-flash excitation of PSII in the S(2) or S(3) state induces processes with time constants around 350ns, which have been assigned previously to energetic relaxation of the oxidized tyrosine (Y(Z)(ox)). Herein we report monitoring of these processes in the time domain of hundreds of nanoseconds by photoacoustic (or 'optoacoustic') experiments involving pressure-wave detection after excitation of PSII membrane particles by ns-laser flashes. We find that specifically for excitation of PSII in the S(2) state, nuclear rearrangements are induced which amount to a contraction of PSII by at least 30Å(3) (time constant of 350ns at 25°C; activation energy of 285+/-50meV). In the S(3) state, the 350-ns-contraction is about 5 times smaller whereas in S(0) and S(1), no volume changes are detectable in this time domain. It is proposed that the classical S(2)=>S(3) transition of the Mn complex is a multi-step process. The first step after Y(Z)(ox) formation involves a fast nuclear rearrangement of the Mn complex and its protein-water environment (~350ns), which may serve a dual role: (1) The Mn- complex entity is prepared for the subsequent proton removal and electron transfer by formation of an intermediate state of specific (but still unknown) atomic structure. (2) Formation of the structural intermediate is associated (necessarily) with energetic relaxation and thus stabilization of Y(Z)(ox) so that energy losses by charge recombination with the Q(A)(-) anion radical are minimized. The intermediate formed within about 350ns after Y(Z)(ox) formation in the S(2)-state is discussed in the context of two recent models of the S(2)=>S(3) transition of the water oxidation cycle. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: From Natural to Artificial.

Energetics and kinetics of photosynthetic water oxidation studied by photothermal beam deflection (PBD) experiments.

Determination of thermodynamic parameters of water oxidation at the photosystem II (PSII) manganese complex is a major challenge. Photothermal beam deflection (PBD) spectroscopy determines enthalpy changes (ΔH) and apparent volume changes which are coupled with electron transfer in the S-state cycle (Krivanek R, Dau H, Haumann M (2008) Biophys J 94: 1890­1903). Recent PBD results on formation of the Q⁻(A)/Y(•+)(Z) radical pair suggest a value of ΔH similar to the free energy change, ΔG, of -540±40 meV previously determined by the analysis of recombination fluorescence, but presently the uncertainty range of ΔH values determined by PBD is still high (±250 meV). In the oxygen-evolving transition, S3−−>S0, the enthalpy change may be close to zero. A prominent non-thermal signal is associated with both Q⁻(A)/Y(•+)(Z) formation (<1 µs) and the S3−−>S0 transition (~1 ms). The observed (apparent) volume expansion (ΔV of about +40 ų per PSII unit) in the S3−−>S0 transition seems to revert, at least partially, the contractions on lower S-transitions and may also comprise contributions from O2 and proton release. The observed volume changes show that the S3−−>S0 transition is accompanied by significant nuclear movements, which likely are of importance with respect to energetics and mechanism of photosynthetic water oxidation. Detailed PBD studies on all S-transitions will contribute to the progress in PSII research by providing insights not accessible by other spectroscopic methods.

The in vivo mechanics of the magnetotactic backbone as revealed by correlative FLIM-FRET and STED microscopy.

Protein interaction and protein imaging strongly benefit from the advancements in time-resolved and superresolution fluorescence microscopic techniques. However, the techniques were typically applied separately and ex vivo because of technical challenges and the absence of suitable fluorescent protein pairs. Here, we show correlative in vivo fluorescence lifetime imaging microscopy Förster resonance energy transfer (FLIM-FRET) and stimulated emission depletion (STED) microscopy to unravel protein mechanics and structure in living cells. We use magnetotactic bacteria as a model system where two proteins, MamJ and MamK, are used to assemble magnetic particles called magnetosomes. The filament polymerizes out of MamK and the magnetosomes are connected via the linker MamJ. Our system reveals that bacterial filamentous structures are more fragile than the connection of biomineralized particles to this filament. More importantly, we anticipate the technique to find wide applicability for the study and quantification of biological processes in living cells and at high resolution.

Diffraction-Unlimited Fluorescence Imaging with an EasySTED Retrofitted Confocal Microscope.

The easySTED technology provides the means to retrofit a confocal microscope to a diffraction-unlimited stimulated emission depletion (STED) microscope.Although commercial STED systems are available today, for many users of confocal laser scanning microscopes the option of retrofitting their confocal system to a STED system ready for diffraction-unlimited imaging may present an attractive option. The easySTED principle allowing for a joint beam path of excitation and depletion light promises some advantages concerning technical complexity and alignment effort for such an STED upgrade. In the one beam path design of easySTED the use of a common laser source, either a supercontinuum source or two separate lasers coupled into the same single-mode fiber, becomes feasible. The alignment of the focal light distribution of the STED beam relative to that of the excitation beam in all three spatial dimensions is therefore omitted respectively reduced to coupling the STED laser into the common single-mode fiber. Thus, only minor modifications need to be applied to the beam path in the confocal microscope to be upgraded. Those comprise adding polarization control elements and the easySTED waveplate, and adapting the beamsplitter to the excitation/STED wavelength combination.

Binary phase masks for easy system alignment and basic aberration sensing with spatial light modulators in STED microscopy.

The use of binary phase patterns to improve the integration and optimization of spatial light modulators (SLM) in an imaging system, especially a confocal microscope, is proposed and demonstrated. The phase masks were designed to create point spread functions (PSF), which exhibit specific sensitivity to major disturbances in the optical system. This allows direct evaluation of misalignment and fundamental aberration modes by simple visual inspection of the focal intensity distribution or by monitoring the central intensity of the PSF. The use of proposed phase masks is investigated in mathematical modelling and experiment for the use in a stimulated emission depletion (STED) microscope applying wavefront shaping by a SLM. We demonstrate the applicability of these phase masks for modal wavefront sensing of low order aberration modes up to the third order of Zernike polynomials, utilizing the point detector of a confocal microscope in a 'guide star' approach. A lateral resolution of ~25 nm is shown in STED imaging of the confocal microscope retrofitted with a SLM and a STED laser and binary phase mask based system optimization.

Seven steps of alternating electron and proton transfer in photosystem II water oxidation traced by time-resolved photothermal beam deflection at improved sensitivity.

The intricate orchestration of electron transfer (ET) and proton transfer (PT) at the Mn4CaOn-cluster of photosystem II (PSII) is mechanistically pivotal but clearly insufficiently understood. Preparations of PSII membrane particles were investigated using a kinetically competent and sensitive method, photothermal beam deflection (PBD), to monitor apparent volume changes of the PSII protein. Driven by nanosecond laser flashes, the PSII was synchronously stepped through its water-oxidation cycle involving four (semi)stable states (S0, S1, S2, and S3) and minimally three additional transiently formed intermediates. The PBD approach was optimized as compared to our previous experiments, resulting in superior signal quality and resolution of more reaction steps. Now seven transitions were detected and attributed, according to the H/D-exchange, temperature, and pH effects on their time constants, to ET or PT events. The ET steps oxidizing the Mn4CaOn cluster in the S2 → S3 and S0 → S1 transitions, a biphasic PT prior to the O2-evolving reaction, as well as the reoxidation of the primary quinone acceptor (QA(-)) at the PSII acceptor side were detected for the first time by PBD. The associated volume changes involve (i) initial formation of charged groups resulting in contraction assignable to electrostriction, (ii) volume contraction explainable by reduced metal-ligand distances upon manganese oxidation, and (iii) charge-compensating proton removal resulting in volume expansion due to electrostriction reversal. These results support a reaction cycle of water oxidation exhibiting alternate ET and PT steps. An extended kinetic scheme for the O2-evolving S3 ⇒ S0 transition is proposed, which includes crucial structural and protonic events.

Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope.

By overcoming the diffraction limit in light microscopy, super-resolution techniques, such as stimulated emission depletion (STED) microscopy, are experiencing an increasing impact on life sciences. High costs and technically demanding setups, however, may still hinder a wider distribution of this innovation in biomedical research laboratories. As far-field microscopy is the most widely employed microscopy modality in the life sciences, upgrading already existing systems seems to be an attractive option for achieving diffraction-unlimited fluorescence microscopy in a cost-effective manner. Here, we demonstrate the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout, previously proposed as "easy-STED", achieving lateral resolution < λ/10 corresponding to a five-fold improvement over a confocal modality. For this purpose, both the excitation and depletion laser beams pass through a commercially available segmented phase plate that creates the STED-doughnut light distribution in the focal plane, while leaving the excitation beam unaltered when implemented into the joint beam path. Diffraction-unlimited imaging of 20 nm-sized fluorescent beads as reference were achieved with the wavelength combination of 635 nm excitation and 766 nm depletion. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and Abberior STAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. Here, we applied a recently proposed deconvolution approach and showed that images obtained from time-gated pulsed STED microscopy may benefit concerning the signal-to-background ratio, from the joint deconvolution of sub-images with different spatial information which were extracted from offline time gating.