Serial femtosecond crystallography (SFX) and continuous serial electron diffraction (c-SerialED) both enable high-resolution structure determination from protein microcrystals with minimal radiation damage, making it ideal for studying redox-active metalloenzymes. Here, c-SerialED and SFX were used to solve structures of the class Ia ribonucleotide reductase R2 subunit in oxidized (FeIII-FeIII), reduced (FeII-FeII), and re-oxidized states at ∼1.8 Å resolution, capturing three points in a redox reaction. These results demonstrate that c-SerialED can track reversible changes at the redox-site, enabling future time-resolved studies. Comparison between c-SerialED structures and SFX diffraction and emission data confirmed minimal radiation damage. Furthermore, previously reported structures use mercury in the crystallization condition and show mercury-induced conformational changes. Here, we use mercury-free crystallization conditions and reveal a water molecule in the redox center of the reduced state, absent in the previous structures, making these structures more representative of the physiological state.

During photosynthetic water oxidation, the Mn4Ca cluster in Photosystem II progresses through five intermediate Si (i = 0-4) states. X-ray crystallography studies have reported the insertion of one new O ligand during the formation of the S3 state, but recent studies question the presence of this additional ligand based on cryo-EM and earlier room-temperature crystallography data. There is also controversy about whether the O-O bond interaction already occurs in the S3 state or in the subsequent S3 to S0 transition. Here we report conventional high-resolution data for the S1, S2, and S3 states to a resolution of ~1.9 Å, and anomalous diffraction data at two energies (9.5 keV and 7 keV), that was used to model the Mn positions, followed by determination of oxygen positions using the high-resolution maps. We show that the new oxygen atom, OX (or O6), in the S3 state is observable as a distinct peak without any restraints, confirming its ligation to Mn1 and Ca. The OX-O5 distance is ~2.1 Å, supporting no strong interaction between them in the S3 state, suggesting that if this is the O-O bond formation site, it is formed during the S3 to S0 transition initiated by the final oxidation of the cluster.

In all photosynthetic reaction centers (RC), transmembrane electron-transfer (ET) cofactor pathways are coordinated by homologous peptides and arranged with approximate C2 symmetry, providing two possible paths for charge separation (designated A and B). In type II RCs-Photosystem II and the purple bacterial RCs-only the A branch is active for ET. A variant bacterial RC containing nine amino acid substitutions that result in high-yield ET along the normally nonfunctional B branch was designed. Structural and theoretical studies were combined to understand factors that control unique ET reactions in the complex. Serial femtosecond crystallography performed at an X-ray Free Electron Laser afforded the room temperature X-ray-damage-free crystal structure of the variant RC, revealing interactions between the substituted amino acids and the ET cofactors. The positions of the bacteriochlorin cofactors on the symmetry-related pathways are unaffected, underscoring that the substitutions alter the energetics of ET, leading to the functional changes. In the binding pockets for the terminal electron acceptor quinones, alterations in the positions of neighboring amino acids and water molecules are coordinated with the change of the substituted side chains. Modifications deactivated A-branch ET and activated transmembrane charge separation along the B branch. Electrostatic calculations based on the structure reveal how differences in stabilization of charge-separated states by the protein environment underlie the change in direction of electron flow in this variant RC and provide insights into mechanisms by which B-branch charge separation across the photosynthetic membrane can be achieved in high yield.

Protein-hydroxylation catalysed by Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases is an important regulatory mechanism in human biology. Such oxygenases typically coordinate their Fe(II) cofactor via a conserved triad of an aspartate- or glutamate- and two histidine-residues. By contrast, aspartate/asparagine β-hydroxylase (AspH), which catalyses asparagine/aspartate-residue oxidation in epidermal growth factor-like domains (EGFDs), has only two histidine-residues (H679, H725), with a water occupying the site normally occupied by an aspartate- or glutamate-residue. We describe mechanistic studies with catalytically active AspH crystals. Turnover studies with single crystals under cryogenic conditions give (3 R)-hydroxylated EGFDs with the product alcohol coordinating Fe(II) trans to H725. Time-resolved serial crystallography of microcrystals using an acoustic droplet ejection system, coupled to X-ray emission analyses, demonstrate turnover within 1.5 s, giving a product complex in which Fe(II) is regenerated. Solution and crystallographic studies with the O2 surrogate nitric oxide imply O2 binds to Fe(II) trans to H725. The additional Fe-chelating water is maintained throughout AspH catalysis and is not directly involved in substrate hydroxylation, because O2 is the sole oxygen source in alcohol products, as shown by 18O labelling studies. The results reveal how AspH accommodates both aspartate- and asparagine-substrates and will assist in efforts targeting AspH for cancer treatment.

The use of X-ray structures to determine and interpret the ferryl iron-oxygen bond order in molecular oxygen-activating heme enzymes has, in the past, been controversial. This has mainly stemmed from the susceptibility of ferryl species to X-ray-induced electronic state changes. In this work we establishe using time-resolved serial femtosecond X-ray crystallography (tr-SFX) on a dye-decolourising peroxidase that the ferryl intermediate species (Compounds I and II) captured following in situ mixing of microcrystals with H2O2 have single, rather than the double bond character expected. X-ray emission validated tr-SFX data with quantum refinement, time-dependent-DFT calculations and QM/MM geometry optimizations together support the concept that the single iron-oxygen bond character is not an indication of ferryl reduction or a protonated form (FeIV-OH) but is instead attributed to the existence of accessible excited states possessing ferric-oxyl (FeIII-O•-) character. Such states offer insight into the nature of ferryl heme.

We report the successful resolution of the multiplet structure of Kα 1 x-ray emission in manganese (Mn) complexes through seeded stimulated x-ray emission spectroscopy (seeded S-XES). Using a femtosecond pump pulse above the Mn K edge to generate simultaneous 1s core holes, and a second-color tunable seed pulse to initiate the stimulated emission process, we were able to enhance individual lines within the Kα 1 emission. This approach allows to resolve the fine multiplet features that are obscured by lifetime broadening in conventional (spontaneous) Mn Kα XES. The work builds on our previous observation that S-XES from Mn(II) and Mn(VII) complexes pumped at high intensities can exhibit stimulated emission without sacrificing the chemical sensitivity to oxidation states. This technique opens the door to controlled high-resolution electronic structure spectroscopy in transition-metal complexes beyond the core-hole lifetime, with potential applications in catalysis, inorganic chemistry, and materials science.

Our work reveals the structure of the active state of Methyl-Coenzyme M Reductase (MCR), the key and rate-limiting enzyme in biological methane formation. We find large differences between the active Ni(I) and inactive Ni(II) proteins and provide insight into how nature makes and breaks the C-H bond of methane. The Ni(II)-F430 center in inactive MCR contains four planar nitrogen ligands, a lower axial glutamine oxo, and an upper axial thiolate. The Ni(I)-enzyme replaces the axial ligands with a single water. The one-electron redox change results in movement of the Ni ion and upward swing of the β-lactam ring in the tetrapyrrole coupled to a domino-like protein quake through second sphere residues, inter-subunit interactions, a substrate tunnel, affecting even the dimensions of the unit cell. These structural changes lead Ni(I)-MCR to release a charge clamp that, in the Ni(II) state, locks down substrate Coenzyme B. Determining the Ni(I)-MCR structure required development of rigorous anaerobic crystallographic techniques. Validation of the MCR redox state was accomplished by in-line and parallel spectroscopic and unit cell analyses. This structure has large implications for developing technologies to limit methane emissions and efficiently produce biofuels. Methodology described here will enhance structural biology for other oxygen-sensitive enzymes. ### Competing Interest Statement The authors have declared no competing interest. Office of Basic Energy SciencesOffice of Basic Energy Sciences, https://ror.org/05mg91w61, DE-FG02-08ER15931, FWP 100593, DE-AC02-05CH11231, DE-SC0014664, DE-AC02-76SF00515, DEAC02-05CH11231 National Institutes of HealthNational Institutes of Health, https://ror.org/01cwqze88, GM149528, GM110501, GM126289, GM117126, GM151988, P30GM133894

Since the invention of the laser, nonlinear effects such as filamentation1, Rabi cycling2,3 and collective emission4 have been explored in the optical regime, leading to a wide range of scientific and industrial applications5-8. X-ray free-electron lasers (XFELs) have extended many optical techniques to X-rays for their advantages of ångström-scale spatial resolution and elemental specificity9. An example is XFEL-driven inner-shell Kα1 (2p3/2 → 1s1/2) X-ray lasing in elements ranging from neon to copper, which has been used for nonlinear spectroscopy and development of new X-ray laser sources10-16. Here we show that strong lasing effects similar to those in the optical regime can occur at 1.5-2.1 Å wavelengths during high-intensity (>1019 W cm-2) XFEL-driven Kα1 lasing of copper and manganese. Depending on the temporal XFEL pump pulse substructure, the resulting X-ray pulses (about 106-108 photons) can exhibit strong spatial inhomogeneities and spectral splitting, inhomogeneities and broadening. Three-dimensional Maxwell-Bloch calculations17 show that the observed spatial inhomogeneities result from X-ray filamentation and that the broad spectral features are driven by sub-femtosecond Rabi cycling. Our simulations indicate that these X-ray pulses can have pulse lengths of less than 100 attoseconds and coherence properties that provide opportunities for quantum X-ray optics applications.

Photosynthesis provides most of the bio-available energy and oxygen to our planet. Yet, while several other mechanisms in oxygenic and anoxygenic photosynthesis have been thoroughly investigated, the core water-splitting reaction of photosystem II (PSII) remains largely uncharacterized. The recent advent of X-ray free-electron lasers has provided us with a tool to probe the structure of PSII's oxygen-evolving complex under ambient conditions and with microsecond time resolution. Still, the quality of diffraction data offered by these experiments is insufficient to reliably observe one-electron differences between individual time points.[1] Instead of probing the imprecise scatterer distribution, oxidation states of individual metal atoms can be assigned by investigating their X-ray absorption edges. Information from classical spectroscopy can not be matched to individual atoms; however, by performing serial diffraction experiments with a pink beam tuned to the metal absorption edge, the anomalous dispersion of each atom becomes embedded in the diffraction image. A careful analysis of Bragg reflection profiles can be thus applied to retrieve the atomic form factors as a function of energy. Refined absorption curves can be then used to characterize the electronic structure of each atom. This spatially resolved anomalous dispersion (SPREAD) technique has been previously successfully applied to data simulated for ferredoxin: a 25 kDa protein containing two differently charged iron centers.[2] The present work describes our recent advances in scaling the pipeline for a 750 kDa PSII with a four-manganese cluster and adapting it to experimental data. In particular, we describe the first working refinement of experimental data, as well as issues encountered with reliability, restraints, mosaicity, and memory use.

Methyl-Coenzyme M Reductase (MCR) catalyzes the forward and reverse reactions of methane biosynthesis and anaerobic methane oxidation, respectively. MCR employs radical chemistry via the Ni-containing cofactor F430, which when in the low Ni(I) valence state leads to the CH3-S bond cleavage of methyl-coenzyme M (CH3SCoM) to generate a methyl radical. The methyl radical abstracts a hydrogen atom from coenzyme-B (HSCoB) to form the heterodisulfide (CoBSSCoM) and the inert, high-energy molecule, methane. Due to MCR’s low redox-potential of the Ni(I)/Ni(II) states, MCR is one of the most oxygen sensitive enzymes on the planet as well as one of the hardest enzymes to harvest and purify in the active-Ni(I) state. Structural biology in MCR has thus been hindered due to MCR’s oxygen sensitivity, and only inhibited, inactive-Ni(II) structures have been solved. Herein, we have solved these gaps of knowledge by elucidating the structure of active-Ni(I) using anaerobic X-ray Free Electron Laser (XFEL) femtosecond serial crystallography and tandem UV-Vis X-ray Diffraction (XRD) utilizing anaerobic capillary counter diffusion. We employ a “diffraction before destruction” principle using XFEL serial crystallography to limit radiation damage to the Ni center to obtain what may be the first Ni(I) active structure revealing a very dynamic structure with high b-factors as well as novel Ni coordination distances. Furthermore, anaerobic counter diffusion protein crystallization was utilized to crystallize proteins inside of an anaerobic capillary used for UV-Vis and XRD to capture the oxidation state once in contact with radiation damage inducing X-rays. This knowledge will further the fields of methane mitigation with an understanding of the active Ni(I) MCR structure and biofuel production by which this enzyme participates in CH functionalization along with the advancement of technology for anaerobic crystallography using MCR as a model for extremely oxygen sensitive enzymes.

We report the successful resolution of the multiplet structure of the Kα1 x-ray emission in manganese (Mn) complexes through seeded stimulated X-ray emission spectroscopy (seeded S-XES). By employing a femtosecond pump pulse above the Mn K edge to generate simultaneous 1s core-holes, and a second-color tunable seed pulse to initiate the stimulated emission process, we were able to enhance individual lines within the Kα1 emission. This approach allows to resolve the fine multiplet features that are obscured by the life-time broadening in conventional Mn Kα XES. The work builds on our previous observation that S-XES from Mn(II) and Mn(VII) complexes pumped at high intensities can exhibit stimulated emission without sacrificing the chemical sensitivity to oxidation states. This technique opens the door to controlled high-resolution electronic structure spectroscopy in transition metal complexes beyond core hole life time broadening with potential applications in catalysis, inorganic chemistry, and materials science.

Soft X-ray absorption spectroscopy of first row transition elements at their respective L-edges provides important information about the oxidation and spin states of the metal centers. However, the associated sample damage in radiation-sensitive samples substantially alters the electronic and chemical structures of redox-active metal centers. Here, we measure the soft X-ray spectrum of the model MnIII(acac)3 complex containing a redox-active MnIII metal center in an octahedral environment with a superconducting transition-edge sensor detector. To reduce the secondary damage resulting primarily from the diffusion of radicals and electrons, the spectra are collected at 30 K and 80 K on solid samples. Starting from the first scan, we detect the contribution of X-ray induced sample damage leading to a change in the MnII intensity. However, at low temperatures, particularly at 30 K, we do not observe a gradual increase in the radiation damage with successive scans with the X-ray beam at the same spot. At our estimated dose of 90 kGy, we find 62% of MnIII(acac)3 is still intact at 30 K. However, at room temperature, we see a gradual increase in radiation damage with increasing numbers of scans at the same spot, which is consistent with the possibility of increased diffusion rates of secondary radicals and electrons as noted in other studies.

X-ray absorption spectroscopy (XAS) of 3d transition metals provides important electronic structure information for many fields. However, X-ray-induced radiation damage under physiological temperature has prevented using this method to study dilute aqueous systems, such as metalloenzymes, as the catalytic reaction proceeds. Here we present a new approach to enable operando XAS of dilute biological samples and demonstrate its feasibility with K-edge XAS spectra from the Mn cluster in photosystem II and the Fe-S centers in photosystem I. This approach combines highly efficient sample delivery strategies and a robust signal normalization method with high-transmission Bragg diffraction-based spectrometers at X-ray free-electron lasers (XFELs) in a damage-free, shot-by-shot mode. These photon-out spectrometers have been optimized for discriminating the metal Mn/Fe Kα fluorescence signals from the overwhelming scattering background present on currently available detectors for XFELs that lack suitable energy discrimination. We quantify the enhanced performance metrics of the spectrometer and discuss its potential applications for acquiring time-resolved XAS spectra of biological samples during their reactions at XFELs.

The photoreaction and commensurate structural changes of a chromophore within biological photoreceptors elicit conformational transitions of the protein promoting the switch between deactivated and activated states. We investigated how this coupling is achieved in a bacterial phytochrome variant, Agp2-PAiRFP2. Contrary to classical protein crystallography, which only allows probing (cryo-trapped) stable states, we have used time-resolved serial femtosecond x-ray crystallography (tr-SFX) and pump-probe techniques with various illumination and delay times with respect to photoexcitation of the parent Pfr state. Thus, structural data for seven time frames were sorted into groups of molecular events along the reaction coordinate. They range from chromophore isomerization to the formation of Meta-F, the intermediate that precedes the functional relevant secondary structure transition of the tongue. Structural data for the early events were used to calculate the photoisomerization pathway to complement the experimental data. Late events allow identifying the molecular switch that is linked to the intramolecular proton transfer as a prerequisite for the following structural transitions.

The water oxidation complex (Mn4CaO5 complex) in Photosystem II is the catalyst for the transformation of two water molecules to molecular oxygen, releasing four electrons and four protons in the process. Starting from the ground state, the complex gets progressively oxidized and moves along the Kok cycle (S0-S4 intermediate states) upon absorption of a photon in each step. In the last state (S4), the complex releases its stored oxidative power to catalyze the reaction and return to the ground state. Here we present our latest results on time-resolved structural studies done at room temperature on Photosystem II using X-ray free-electron laser (XFEL) crystallography. In particular, we will discuss findings from the final S3 -S0 reaction where molecular oxygen is formed. The data reveals well-coordinated structural changes in the active site and the broader protein environment to catalyze the reaction and give us clues to the possible reaction pathway. We will also discuss recent work using crystalline molecular dynamics to interpret water structure in the XFEL data.

Kenneth (Ken) Sauer was a mainstay of research in photosynthesis at the University of California, Berkeley and the Lawrence Berkeley National Laboratory (LBNL) for more than 50 years. Ken will be remembered by his colleagues, and other workers in the field of photosynthesis as well, for his pioneering work that introduced the physical techniques whose application have enriched our understanding of the basic reactions of oxygenic photosynthesis. His laboratory was a training ground for many students and postdocs who went on to success in the field of photosynthesis and many others. Trained as a physical chemist, he always brought that quantitative approach to research questions and used several spectroscopic methods in his research. His broad scientific interests concerned the role of manganese in oxygen evolution, electronic properties of chlorophylls, energy transport in antenna complexes, and electron transport reactions. He was also an enthusiastic teacher, an enormously successful mentor who leaves behind a legion of scientists as his abiding legacy, a lover of music and the outdoors with many interests beyond science, and a dedicated family man with a great sense of humility. In this tribute, we summarize some aspects of Ken Sauer’s life and career, illustrated with selected research achievements, and describe his approach to research and life as we perceived it, which is complemented by reminiscences of several current researchers in photosynthesis and other fields. The supporting material includes Ken Sauers’s CV and publication list, as well as a list of the graduate students and postdocs he trained and of researchers that spent a sabbatical in his lab.

In natural photosynthesis, the light-driven splitting of water into electrons, protons and molecular oxygen forms the first step of the solar-to-chemical energy conversion process. The reaction takes place in photosystem II, where the Mn 4 CaO 5 cluster first stores four oxidizing equivalents, the S 0 to S 4 intermediate states in the Kok cycle, sequentially generated by photochemical charge separations in the reaction center and then catalyzes the O–O bond formation chemistry 1 – 3 . Here, we report room temperature snapshots by serial femtosecond X-ray crystallography to provide structural insights into the final reaction step of Kok’s photosynthetic water oxidation cycle, the S 3 →[S 4 ]→S 0 transition where O 2 is formed and Kok’s water oxidation clock is reset. Our data reveal a complex sequence of events, which occur over micro- to milliseconds, comprising changes at the Mn 4 CaO 5 cluster, its ligands and water pathways as well as controlled proton release through the hydrogen-bonding network of the Cl1 channel. Importantly, the extra O atom O x , which was introduced as a bridging ligand between Ca and Mn1 during the S 2 →S 3 transition 4 – 6 , disappears or relocates in parallel with Y z reduction starting at approximately 700 μs after the third flash. The onset of O 2 evolution, as indicated by the shortening of the Mn1–Mn4 distance, occurs at around 1,200 μs, signifying the presence of a reduced intermediate, possibly a bound peroxide.

Photosynthesis-the conversion of energy from sunlight into chemical energy-is essential for life on Earth. Yet there is much we do not understand about photosynthetic energy conversion on a fundamental level: how it evolved and the extent of its diversity, its dynamics, and all the components and connections involved in its regulation. In this commentary, researchers working on fundamental aspects of photosynthesis including the light-dependent reactions, photorespiration, and C4 photosynthetic metabolism pose and discuss what they view as the most compelling open questions in their areas of research.