Inspired by the period-four oscillation in flash-induced oxygen evolution of photosystem II discovered by Joliot in 1969, Kok performed additional experiments and proposed a five-state kinetic model for photosynthetic oxygen evolution, known as Kok’s S-state clock or cycle1,2. The model comprises four (meta)stable intermediates (S0, S1, S2 and S3) and one transient S4 state, which precedes dioxygen formation occurring in a concerted reaction from two water-derived oxygens bound at an oxo-bridged tetra manganese calcium (Mn4CaO5) cluster in the oxygen-evolving complex3–7. This reaction is coupled to the two-step reduction and protonation of the mobile plastoquinone QB at the acceptor side of PSII. Here, using serial femtosecond X-ray crystallography and simultaneous X-ray emission spectroscopy with multi-flash visible laser excitation at room temperature, we visualize all (meta)stable states of Kok’s cycle as high-resolution structures (2.04–2.08 Å). In addition, we report structures of two transient states at 150 and 400 μs, revealing notable structural changes including the binding of one additional ‘water’, Ox, during the S2→S3 state transition. Our results suggest that one water ligand to calcium (W3) is directly involved in substrate delivery. The binding of the additional oxygen Ox in the S3 state between Ca and Mn1 supports O–O bond formation mechanisms involving O5 as one substrate, where Ox is either the other substrate oxygen or is perfectly positioned to refill the O5 position during O2 release. Thus, our results exclude peroxo-bond formation in the S3 state, and the nucleophilic attack of W3 onto W2 is unlikely.

Light-induced oxidation of water by photosystem II (PS II) in plants, algae and cyanobacteria has generated most of the dioxygen in the atmosphere. PS II, a membrane-bound multi-subunit pigment-protein complex, couples the one-electron photochemistry at the reaction center with the four-electron redox chemistry of water oxidation at the Mn4CaO5 cluster in the oxygen-evolving complex (OEC) (Fig. 1a, Extended Data Fig. 1). Under illumination, the OEC cycles through five intermediate S-states (S0 to S4)1, where S1 is the dark stable state and S3 is the last semi-stable state before O-O bond formation and O2 evolution2,3. A detailed understanding of the O-O bond formation mechanism remains a challenge, and elucidating the structures of the OEC in the different S-states, as well as the binding of the two substrate waters to the catalytic site4-6, is a prerequisite for this purpose. Here we report the use of femtosecond pulses from an X-ray free electron laser (XFEL) to obtain damage free, room temperature (RT) structures of dark-adapted (S1), two-flash illuminated (2F; S3-enriched), and ammonia-bound two-flash illuminated (2F-NH3; S3-enriched) PS II. Although the recent 1.95 Å structure of PS II7 at cryogenic temperature using an XFEL provided a damage-free view of the S1 state, RT measurements are required to study the structural landscape of proteins under functional conditions8,9, and also for in situ advancement of the S-states. To investigate the water-binding site(s), ammonia, a water analog, has been used as a marker, as it binds to the Mn4CaO5 cluster in the S2 and S3 states10. Since the ammonia-bound OEC is active, the ammonia-binding Mn site is not a substrate water site10-13. Thus, this approach, together with a comparison of the native dark and 2F states, is used to discriminate between proposed O-O bond formation mechanisms.

Phytochromes constitute a class of photoreceptors that can be photoconverted between two stable states. The tetrapyrrole chromophore absorbs in the red spectral region and displays fluorescence maxima above 700 nm, albeit with low quantum yields. Because this wavelength region is particularly advantageous for fluorescence-based deep tissue imaging, there is a strong interest to engineer phytochrome variants with increased fluorescence yields. Such targeted design efforts would substantially benefit from a deeper understanding of those structural parameters that control the photophysical properties of the protein-bound chromophore. Here we have employed resonance Raman (RR) spectroscopy and molecular dynamics simulations for elucidating the chromophore structural changes in a fluorescence-optimized mutant (iRFP) derived from the PAS-GAF domain of the bacteriophytochrome RpBphP2 from Rhodopseudomas palustris . Both methods consistently reveal the structural consequences of the amino acid substitutions in the vicinity of the biliverdin chromophore that may account for lowering the propability of nonradiative excited state decays. First, compared to the wild-type protein, the tilt angle of the terminal ring D with respect to ring C is increased in iRFP, accompanied by the loss of hydrogen bond interactions of the ring D carbonyl function and the reduction of the number of water molecules in that part of the chromophore pocket. Second, the overall flexibility of the chromophore is significantly reduced, particularly in the region of rings D and A, thereby reducing the conformational heterogeneity of the methine bridge between rings A and B and the ring A carbonyl group, as concluded from the RR spectra of the wild-type proteins.

A lysine residue within the highly conserved center of the fifth transmembrane segment in P IIC -type ATPase ␣-subunits is uniquely found in H,K-ATPases instead of a serine in all Na,K-ATPase isoforms. Because previous studies suggested a prominent role of this residue in determining the electrogenicity of non-gastric H,K-ATPase and in pK a modulation of the proton-translocating residues in the gastric H,K-ATPases as well, we investigated its functional significance for ion transport by expressing several Lys-791 variants of the gastric H,KATPase in Xenopus oocytes. Although the mutant proteins were all detected at the cell surface, none of the investigated mutants displayed any measurable K ؉ -induced stationary currents. In Rb ؉ uptake measurements, replacement of Lys-791by Arg, Ala, Ser, and Glu substantially impaired transport activity and reduced the sensitivity toward the E 2 -specific inhibitor SCH28080. Furthermore, voltage clamp fluorometry using a reporter site in the TM5/TM6 loop for labeling with tetramethylrhodamine-6-maleimide revealed markedly changed fluorescence signals. All four investigated mutants exhibited a strong shift toward the E 1 P state, in agreement with their reduced SCH28080 sensitivity, and an about 5-10-fold decreased forward rate constant of the E 1 P 7 E 2 P conformational transition, thus explaining the E 1 P shift and the reduced Rb ؉ transport activity. When Glu-820 in TM6 adjacent to Lys-791 was replaced by non-charged or positively charged amino acids, severe effects on fluorescence signals and Rb ؉ transport were also observed, whereas substitution by aspartate was less disturbing. These results suggest that formation of an E 2 Pstabilizing interhelical salt bridge is essential to prevent futile proton exchange cycles of H ؉ pumping P-type ATPases.

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