當舎 武彦

Animal-like cryptochromes are photoreceptors that control circadian rhythm and signaling in many eukaryotes. Transient photoreduction of the cryptochrome flavin chromophore initiated signaling via a poorly understood mechanism. By serial femtosecond crystallography (SFX), we show that the photoreduction mechanism of Chlamydomonas reinhardtii cryptochrome involves three loci [carboxyl-terminal region, a transient protonation pathway, and flavin adenine dinucleotide (FAD)-binding site] acting in unison to accomplish three effects: radical pair stabilization, protonation of FAD radical, and formation of the signaling state. Using 19 time-resolved SFX snapshots between 10 nanoseconds and 233 milliseconds, we found that light-driven FAD•-/tyrosyl-373 radical pair (RP) formation primes α22 unfolding. Electron transfer-dependent protonation of aspartate-321 by tyrosine-373 is the epicenter of unfolding by disrupting salt bridges between α22 and the photolyase homology region. Before helix unfolding, another pathway opens transiently for FAD•- protonation and RP stabilization. This link between RP formation and conformational changes provides a structural basis for signaling by animal-like cryptochromes.

Photolyases repair UV damage to DNA by using absorbed blue light. Within the photolyase/cryptochrome superfamily (PCSf), a major subgroup consists of prokaryotic (6-4) photolyases. These enzymes rely on flavin adenine dinucleotide (FAD) as a catalytic cofactor, besides an ancillary antenna chromophore, and a [4Fe-4S] cluster with yet unknown function. For the prokaryotic 6-4 photolyase of Caulobacter crescentus, we investigated structural changes associated with its different redox states by damage-free crystallography using X-ray free-electron lasers. EPR and optical spectroscopy confirmed redox-dependent structural transitions, including the formation of an oxidized [4Fe-4S]3+ cluster with the dynamic cleavage of a single iron-sulfur bond. Photoreduction to the catalytic FADH- state alters the flavin binding site at the proximal aromatic pair Y390/F394 that is part of the electron transport pathway. Upon oxidation, the observable structural transitions of the protein matrix around the [4Fe-4S] cluster may affect DNA binding and are consistent with the much-debated role of the iron-sulfur cluster in DNA-binding proteins for quenching electron holes.

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.

Photosystem II (PSII) catalyses the oxidation of water through a four-step cycle of Si states (i = 0-4) at the Mn4CaO5 cluster1-3, during which an extra oxygen (O6) is incorporated at the S3 state to form a possible dioxygen4-7. Structural changes of the metal cluster and its environment during the S-state transitions have been studied on the microsecond timescale. Here we use pump-probe serial femtosecond crystallography to reveal the structural dynamics of PSII from nanoseconds to milliseconds after illumination with one flash (1F) or two flashes (2F). YZ, a tyrosine residue that connects the reaction centre P680 and the Mn4CaO5 cluster, showed structural changes on a nanosecond timescale, as did its surrounding amino acid residues and water molecules, reflecting the fast transfer of electrons and protons after flash illumination. Notably, one water molecule emerged in the vicinity of Glu189 of the D1 subunit of PSII (D1-E189), and was bound to the Ca2+ ion on a sub-microsecond timescale after 2F illumination. This water molecule disappeared later with the concomitant increase of O6, suggesting that it is the origin of O6. We also observed concerted movements of water molecules in the O1, O4 and Cl-1 channels and their surrounding amino acid residues to complete the sequence of electron transfer, proton release and substrate water delivery. These results provide crucial insights into the structural dynamics of PSII during S-state transitions as well as O-O bond formation.

Cytochrome c oxidase (CcO) is part of the respiratory chain and contributes to the electrochemical membrane gradient in mitochondria as well as in many bacteria, as it uses the energy released in the reduction of oxygen to pump protons across an energy-transducing biological membrane. Here, we use time-resolved serial femtosecond crystallography to study the structural response of the active site upon flash photolysis of carbon monoxide (CO) from the reduced heme a3 of ba3-type CcO. In contrast with the aa3-type enzyme, our data show how CO is stabilized on CuB through interactions with a transiently ordered water molecule. These results offer a structural explanation for the extended lifetime of the CuB-CO complex in ba3-type CcO and, by extension, the extremely high oxygen affinity of the enzyme.

Characterization of short-lived reaction intermediates is essential for elucidating the mechanism of the reaction catalyzed by metalloenzymes. Here, we demonstrated that the photolysis of a caged compound under cryogenic temperature followed by thermal annealing is an invaluable technique for trapping of short-lived reaction intermediates of metalloenzymes through the study of membrane-integrated nitric oxide reductase (NOR) that catalyzes reductive coupling of two NO molecules to N2O at its heme/nonheme FeB binuclear center. Although NO produced by the photolysis of caged NO did not react with NOR under cryogenic temperature, annealing to ∼160 K allowed NO to diffuse and react with NOR, which was evident from the appearance of EPR signals assignable to the S = 3/2 state. This indicates that the nonheme FeB-NO species can be trapped as the intermediate. Time-resolved IR spectroscopy with the use of the photolysis of caged NO as a reaction trigger showed that the intermediate formed at 10 μs gave the NO stretching frequency at 1683 cm-1 typical of nonheme Fe-NO, confirming that the combination of the cryo-photolysis of caged NO and annealing enabled us to trap the reaction intermediate. Thus, the cryo-photolysis of the caged compound has great potential for the characterization of short-lived reaction intermediates.

Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to rise and would become untreatable. The development of antibiotics with a different mechanism of action is seriously required. Here, we identified an allosteric inhibitory site buried inside eukaryotic mitochondrial heme-copper oxidases (HCOs), the essential respiratory enzymes for life. The steric conformation around the binding pocket of HCOs is highly conserved among bacteria and eukaryotes, yet the latter has an extra helix. This structural difference in the conserved allostery enabled us to rationally identify bacterial HCO-specific inhibitors: an antibiotic compound against ceftriaxone-resistant Neisseria gonorrhoeae. Molecular dynamics combined with resonance Raman spectroscopy and stopped-flow spectroscopy revealed an allosteric obstruction in the substrate accessing channel as a mechanism of inhibition. Our approach opens fresh avenues in modulating protein functions and broadens our options to overcome AMR.

Controlling the reactivity of high-valent Fe(IV)-O catalytic intermediates, Compounds I and II, generated in heme enzymes upon reaction with dioxygen or hydrogen peroxide, is important for function. It has been hypothesized that the presence (wet) or absence (dry) of distal heme pocket water molecules can influence whether Compound I undergoes sequential one-electron additions or a concerted two-electron reduction. To test this hypothesis, we investigate the role of water in the heme distal pocket of a dye-decolorizing peroxidase utilizing a combination of serial femtosecond crystallography and rapid kinetic studies. In a dry distal heme site, Compound I reduction proceeds through a mechanism in which Compound II concentration is low. This reaction shows a strong deuterium isotope effect, indicating that reduction is coupled to proton uptake. The resulting protonated Compound II (Fe(IV)-OH) rapidly reduces to the ferric state, giving the appearance of a two-electron transfer process. In a wet site, reduction of Compound I is faster, has no deuterium effect, and yields highly populated Compound II, which is subsequently reduced to the ferric form. This work provides a definitive experimental test of the hypothesis advanced in the literature that relates sequential or concerted electron transfer to Compound I in wet or dry distal heme sites.

Abstract The enzymatic recycling of polyethylene terephthalate (PET) can be a promising approach to tackle the problem of plastic waste. The thermostability and activity of PET-hydrolyzing enzymes are still insufficient for practical application. Pretreatment of PET waste is needed for bio-recycling. Here, we analyzed the degradation of PET films, packages, and bottles using the newly engineered cutinase Cut190. Using gel permeation chromatography and high-performance liquid chromatography, the degradation of PET films by the Cut190 variant was shown to proceed via a repeating two-step hydrolysis process; initial endo-type scission of a surface polymer chain, followed by exo-type hydrolysis to produce mono/bis(2-hydroxyethyl) terephthalate and terephthalate from the ends of fragmented polymer molecules. Amorphous PET powders were degraded more than twofold higher than amorphous PET film with the same weight. Moreover, homogenization of post-consumer PET products, such as packages and bottles, increased their degradability, indicating the importance of surface area for the enzymatic hydrolysis of PET. In addition, it was required to maintain an alkaline pH to enable continuous enzymatic hydrolysis, by increasing the buffer concentration (HEPES, pH 9.0) depending on the level of the acidic products formed. The cationic surfactant dodecyltrimethylammonium chloride promoted PET degradation via adsorption on the PET surface and binding to the anionic surface of the Cut190 variant. The Cut190 variant also hydrolyzed polyethylene furanoate. Using the best performing Cut190 variant (L136F/Q138A/S226P/R228S/D250C-E296C/Q123H/N202H/K305del/L306del/N307del) and amorphous PET powders, more than 90 mM degradation products were obtained in 3 days and approximately 80 mM in 1 day. Graphical Abstract

Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.

Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyze the reduction of nitrite to nitric oxide (NO), have proven to be a good model system for studying these complex processes including proton-coupled electron transfer (ET) and their orchestration for substrate binding/utilization. Recently, a two-domain CuNiR from a Rhizobia species (Br2DNiR) has been discovered with a substantially lower enzymatic activity where the catalytic type-2 Cu (T2Cu) site is occupied by two water molecules requiring their displacement for the substrate nitrite to bind. Single crystal spectroscopy combined with MSOX (multiple structures from one crystal) for both the as-isolated and nitrite-soaked crystals clearly demonstrate that inter-Cu ET within the coupled T1Cu-T2Cu redox system is heavily gated. Laser-flash photolysis and optical spectroscopy showed rapid ET from photoexcited NADH to the T1Cu center but little or no inter-Cu ET in the absence of nitrite. Furthermore, incomplete reoxidation of the T1Cu site (∼20% electrons transferred) was observed in the presence of nitrite, consistent with a slow formation of NO species in the serial structures of the MSOX movie obtained from the nitrite-soaked crystal, which is likely to be responsible for the lower activity of this CuNiR. Our approach is of direct relevance for studying redox reactions in a wide range of biological systems including metalloproteins that make up at least 30% of all proteins.

Bacterial pathogens acquire heme from the host hemoglobin as an iron nutrient for their virulence and proliferation in blood. Concurrently, they encounter cytotoxic-free heme that escapes the heme-acquisition process. To overcome this toxicity, many gram-positive bacteria employ an ATP-binding cassette heme-dedicated efflux pump, HrtBA in the cytoplasmic membranes. Although genetic analyses have suggested that HrtBA expels heme from the bacterial membranes, the molecular mechanism of heme efflux remains elusive due to the lack of protein studies. Here, we show the biochemical properties and crystal structures of Corynebacterium diphtheriae HrtBA, alone and in complex with heme or an ATP analog, and we reveal how HrtBA extracts heme from the membrane and releases it. HrtBA consists of two cytoplasmic HrtA ATPase subunits and two transmembrane HrtB permease subunits. A heme-binding site is formed in the HrtB dimer and is laterally accessible to heme in the outer leaflet of the membrane. The heme-binding site captures heme from the membrane using a glutamate residue of either subunit as an axial ligand and sequesters the heme within the rearranged transmembrane helix bundle. By ATP-driven HrtA dimerization, the heme-binding site is squeezed to extrude the bound heme. The mechanism sheds light on the detoxification of membrane-bound heme in this bacterium.

Steady-state kinetics of cytochrome-c dependent denitrifying NO reductases (cNORs) show evidence of substrate inhibition at NO concentrations higher than 10 μM, but the mechanism of inhibition remains unclear. Here, we present low-temperature FTIR photolysis experiments carried out on the NO complex formed by addition of NO to the oxidized cNORs. A differential signal at 1261 cm-1 that downshifts with 15NO and 15N18O is assigned to a ν(NO2) from a bridging diiron-nitrito complex at the heme-nonheme diron site. Theoretical calculations reproduces observed frequencies and isotope shifts. Our experimental results confirm a prior theoretical study by Blomberg and Siegbahn [Blomberg, M. R., and Siegbahn, P. E. M. Biochemistry 2012, 51, 5173-5186] that proposed substrate inhibition through a radical combination reaction between the diferric μ-oxo group and an NO molecule to form a heme Fe(III)-nitrito-FeB(II) inhibitory complex. Stopped-flow experiments suggest that substrate inhibition also occurs after a half-reduction cycle, i.e. when fully-reduced cNOR reduces two NO molecules at the heme-nonheme diferrous active site cluster to produce one N2O molecule and the diferric cluster. These results support catalytic mechanisms that proceed through isomerization of a diferric-hyponitrite transient complex to produce a bridging diferric μ-oxo group and N2O without protonation of the putative hyponitrite intermediate.

Elucidation of molecular mechanism for metalloeznyme-catalyzed reactions is longstanding topics in the bio-inorganic chemistry field, since metalloeznymes play pivotal roles in various biological processes and catalyze reactions with high efficiency under mild conditions. Structural determination of reaction intermediates is key to visualizing the reactions catalyzed by metalloenzymes. Although it is not easy to determine the structure of the transient species by conventional crystallography, newly developed time-resolved X-ray crystallography using X-ray free electron laser (XFEL) has a great potential for the structural characterization of intermediates. However, XFEL-based time-resolved crystallography requires a photo-trigger, hampering its application to non-photosensitive proteins like metalloenzymes. Here, to overcome this issue, we focused on caged substrates, which produce the substrate upon photo-irradiation, to introduce the photo-trigger into the reaction catalyzed by the metalloenzyme. To demonstrate the usefulness of caged substrates for the trigger of XFEL-based time-resolved crystallography, soluble nitric oxide (NO) reductase, which catalyzes the reduction of NO to nitrous oxide (N2O) at a heme active center, and caged NO were used as a model system. Time-resolved spectroscopic analysis showed that the photolysis of caged NO could initiate NO reduction by P450nor in the micro-crystals. Time-resolved crystallography using XFEL enabled us to determine the structures of two intermediates; NO-bound form and subsequent NO-activated form, which provided a unique opportunity to draw the complete picture of the reaction cycle of P450nor. Thus, the combination of caged substrate and XFEL-based time-resolved crystallography is an invaluable method for the visualization of the reactions catalyzed by metalloenzyems.

Oxygen activation in all heme enzymes requires the formation of high oxidation states of iron, usually referred to as ferryl heme. There are two known intermediates: Compound I and Compound II. The nature of the ferryl heme-and whether it is an FeIV =O or FeIV -OH species-is important for controlling reactivity across groups of heme enzymes. The most recent evidence for Compound I indicates that the ferryl heme is an unprotonated FeIV =O species. For Compound II, the nature of the ferryl heme is not unambiguously established. Here, we report 1.06 Å and 1.50 Å crystal structures for Compound II intermediates in cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), collected using the X-ray free electron laser at SACLA. The structures reveal differences between the two peroxidases. The iron-oxygen bond length in CcP (1.76 Å) is notably shorter than in APX (1.87 Å). The results indicate that the ferryl species is finely tuned across Compound I and Compound II species in closely related peroxidase enzymes. We propose that this fine-tuning is linked to the functional need for proton delivery to the heme.

Nitric oxide (NO) reductase from the fungus Fusarium oxysporum is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide (N2O) in the global nitrogen cycle. In this enzymatic reaction, the heme-bound NO is activated by the direct hydride transfer from NADH to generate a short-lived intermediate ( I ), a key state to promote N-N bond formation and N-O bond cleavage. This study applied time-resolved (TR) techniques in conjunction with photolabile-caged NO to gain direct experimental results for the characterization of the coordination and electronic structures of I TR freeze-trap crystallography using an X-ray free electron laser (XFEL) reveals highly bent Fe-NO coordination in I , with an elongated Fe-NO bond length (Fe-NO = 1.91 Å, Fe-N-O = 138°) in the absence of NAD+ TR-infrared (IR) spectroscopy detects the formation of I with an N-O stretching frequency of 1,290 cm-1 upon hydride transfer from NADH to the Fe3+-NO enzyme via the dissociation of NAD+ from a transient state, with an N-O stretching of 1,330 cm-1 and a lifetime of ca. 16 ms. Quantum mechanics/molecular mechanics calculations, based on these crystallographic and IR spectroscopic results, demonstrate that the electronic structure of I is characterized by a singly protonated Fe3+-NHO•- radical. The current findings provide conclusive evidence for the N2O generation mechanism via a radical-radical coupling of the heme nitroxyl complex with the second NO molecule.

Hemes (iron-porphyrins) are critical for biological processes in all organisms. Hemolytic bacteria survive by acquiring b-type heme from hemoglobin in red blood cells from their animal hosts. These bacteria avoid the cytotoxicity of excess heme during hemolysis by expressing heme-responsive sensor proteins that act as transcriptional factors to regulate the heme efflux system in response to the cellular heme concentration. Here, the underlying regulatory mechanisms were investigated using crystallographic, spectroscopic, and biochemical studies to understand the structural basis of the heme-responsive sensor protein PefR from Streptococcus agalactiae, a causative agent of neonatal life-threatening infections. Structural comparison of heme-free PefR, its complex with a target DNA, and heme-bound PefR revealed that unique heme coordination controls a >20 Å structural rearrangement of the DNA binding domains to dissociate PefR from the target DNA. We also found heme-bound PefR stably binds exogenous ligands, including carbon monoxide, a by-product of the heme degradation reaction.

Copper-containing nitrite reductases (CuNiRs), encoded by nirK gene, are found in all kingdoms of life with only 5% of CuNiR denitrifiers having two or more copies of nirK Recently, we have identified two copies of nirK genes in several α-proteobacteria of the order Rhizobiales including Bradyrhizobium sp. ORS 375, encoding a four-domain heme-CuNiR and the usual two-domain CuNiR (Br2DNiR). Compared with two of the best-studied two-domain CuNiRs represented by the blue (AxNiR) and green (AcNiR) subclasses, Br2DNiR, a blue CuNiR, shows a substantially lower catalytic efficiency despite a sequence identity of ~70%. Advanced synchrotron radiation and x-ray free-electron laser are used to obtain the most accurate (atomic resolution with unrestrained SHELX refinement) and damage-free (free from radiation-induced chemistry) structures, in as-isolated, substrate-bound, and product-bound states. This combination has shed light on the protonation states of essential catalytic residues, additional reaction intermediates, and how catalytic efficiency is modulated.

Obtaining structures of intact redox states of metal centers derived from zero dose X-ray crystallography can advance our mechanistic understanding of metalloenzymes. In dye-decolorising heme peroxidases (DyPs), controversy exists regarding the mechanistic role of the distal heme residues aspartate and arginine in the heterolysis of peroxide to form the catalytic intermediate compound I (FeIV =O and a porphyrin cation radical). Using serial femtosecond X-ray crystallography (SFX), we have determined the pristine structures of the FeIII and FeIV =O redox states of a B-type DyP. These structures reveal a water-free distal heme site that, together with the presence of an asparagine, imply the use of the distal arginine as a catalytic base. A combination of mutagenesis and kinetic studies corroborate such a role. Our SFX approach thus provides unique insight into how the distal heme site of DyPs can be tuned to select aspartate or arginine for the rate enhancement of peroxide heterolysis.

<italic>Neisseria meningitidis</italic> is carried by nearly a billion humans, causing developmental impairment and over 100 000 deaths a year. A quinol-dependent nitric oxide reductase (qNOR) plays a critical role in the survival of the bacterium in the human host. X-ray crystallographic analyses of qNOR, including that from <italic>N. meningitidis</italic> (<italic>Nm</italic>qNOR) reported here at 3.15 Å resolution, show monomeric assemblies, despite the more active dimeric sample being used for crystallization. Cryo-electron microscopic analysis of the same chromatographic fraction of <italic>Nm</italic>qNOR, however, revealed a dimeric assembly at 3.06 Å resolution. It is shown that zinc (which is used in crystallization) binding near the dimer-stabilizing TMII region contributes to the disruption of the dimer. A similar destabilization is observed in the monomeric (∼85 kDa) cryo-EM structure of a mutant (Glu494Ala) qNOR from the opportunistic pathogen <italic>Alcaligenes</italic> (<italic>Achromobacter</italic>) <italic>xylosoxidans</italic>, which primarily migrates as a monomer. The monomer–dimer transition of qNORs seen in the cryo-EM and crystallographic structures has wider implications for structural studies of multimeric membrane proteins. X-ray crystallographic and cryo-EM structural analyses have been performed on the same chromatographic fraction of <italic>Nm</italic>qNOR to high resolution. This represents one of the first examples in which the two approaches have been used to reveal a monomeric assembly <italic>in crystallo</italic> and a dimeric assembly in vitrified cryo-EM grids. A number of factors have been identified that may trigger the destabilization of helices that are necessary to preserve the integrity of the dimer. These include zinc binding near the entry of the putative proton-transfer channel and the preservation of the conformational integrity of the active site. The mutation near the active site results in disruption of the active site, causing an additional destabilization of helices (TMIX and TMX) that flank the proton-transfer channel helices, creating an inert monomeric enzyme.

BACKGROUND: The invention of the X-ray free-electron laser (XFEL) has provided unprecedented new opportunities for structural biology. The advantage of XFEL is an intense pulse of X-rays and a very short pulse duration (<10 fs) promising a damage-free and time-resolved crystallography approach. SCOPE OF REVIEW: Recent time-resolved crystallographic analyses in XFEL facility SACLA are reviewed. Specifically, metalloproteins involved in the essential reactions of bioenergy conversion including photosystem II, cytochrome c oxidase and nitric oxide reductase are described. MAJOR CONCLUSIONS: XFEL with pump-probe techniques successfully visualized the process of the reaction and the dynamics of a protein. Since the active center of metalloproteins is very sensitive to the X-ray radiation, damage-free structures obtained by XFEL are essential to draw mechanistic conclusions. Methods and tools for sample delivery and reaction initiation are key for successful measurement of the time-resolved data. GENERAL SIGNIFICANCE: XFEL is at the center of approaches to gain insight into complex mechanism of structural dynamics and the reactions catalyzed by biological macromolecules. Further development has been carried out to expand the application of time-resolved X-ray crystallography. This article is part of a Special Issue entitled Novel measurement techniques for visualizing 'live' protein molecules.