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Instruments / Technical Area	Publication Details	Published
I24-Microfocus Macromolecular Crystallography	Serial femtosecond crystallography reveals the role of water in the one- or two-electron redox chemistry of compound I in the catalytic cycle of the B-type dye-decolorizing peroxidase DtpB Marina Lucic , Matthew T. Wilson , Takehiko Tosha , Hiroshi Sugimoto , Anastasiia Shilova , Danny Axford , Robin L. Owen , Michael A. Hough , Jonathan A. R. Worrall Acs Catalysis 26, 13349 - 13359 DOI: 10.1021/acscatal.2c03754 Diamond Proposal Number(s): [25108, 28583] Open Access Show Abstract ▼ Abstract: 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.	Oct 2022
I24-Microfocus Macromolecular Crystallography	Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature Tadeo Moreno-Chicano , Leiah M. Carey , Danny Axford , John H. Beale , R. Bruce Doak , Helen M. E. Duyvesteyn , Ali Ebrahim , Robert W. Henning , Diana C. F. Monteiro , Dean A. Myles , Shigeki Owada , Darren A. Sherrell , Megan L. Straw , Vukica Šrajer , Hiroshi Sugimoto , Kensuke Tono , Takehiko Tosha , Ivo Tews , Martin Trebbin , Richard W. Strange , Kevin L. Weiss , Jonathan A. R. Worrall , Flora Meilleur , Robin L. Owen , Reza A. Ghiladi , Michael A. Hough Iucrj 9 DOI: 10.1107/S2052252522006418 Diamond Proposal Number(s): [14493] Open Access Show Abstract ▼ 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.	Sep 2022
I24-Microfocus Macromolecular Crystallography	Single crystal spectroscopy and multiple structures from one crystal (MSOX) define catalysis in copper nitrite reductases Samuel L. Rose , Seiki Baba , Hideo Okumura , Svetlana V. Antonyuk , Daisuke Sasaki , Tobias M. Hedison , Muralidharan Shanmugam , Derren J. Heyes , Nigel S. Scrutton , Takashi Kumasaka , Takehiko Tosha , Robert R. Eady , Masaki Yamamoto , S. Samar Hasnain Proceedings Of The National Academy Of Sciences 119 DOI: 10.1073/pnas.2205664119 Open Access Show Abstract ▼ Abstract: 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.	Jul 2022
	XFEL crystal structures of peroxidase compound II Hanna Kwon , Jaswir Basran , Chinar Pathak , Mahdi Hussain , Samuel L. Freeman , Alistair J. Fielding , Anna J. Bailey , Natalia Stefanou , Hazel A. Sparkes , Takehiko Tosha , Keitaro Yamashita , Kunio Hirata , Hironori Murakami , Go Ueno , Hideo Ago , Kensuke Tono , Masaki Yamamoto , Hitomi Sawai , Yoshitsugu Shiro , Hiroshi Sugimoto , Emma Raven , Peter C. E. Moody Angewandte Chemie International Edition DOI: 10.1002/anie.202103010 Open Access Show Abstract ▼ Abstract: 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 Fe IV =O or Fe IV ‐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 Fe IV =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 (C c P) 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 C c P (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.	Apr 2021
I03-Macromolecular Crystallography	An unprecedented insight into the catalytic mechanism of copper nitrite reductase from atomic-resolution and damage-free structures Samuel L. Rose , Svetlana V. Antonyuk , Daisuke Sasaki , Keitaro Yamashita , Kunio Hirata , Go Ueno , Hideo Ago , Robert R. Eady , Takehiko Tosha , Masaki Yamamoto , S. Samar Hasnain Science Advances 7 DOI: 10.1126/sciadv.abd8523 Diamond Proposal Number(s): [15991] Open Access Show Abstract ▼ Abstract: 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.	Jan 2021
	Serial femtosecond zero dose crystallography captures a water‐free distal heme site in a dye‐decolourising peroxidase to reveal a catalytic role for an arginine in FeIV=O formation Marina Lucic , Dimitri Svistunenko , Michael Wilson , Amanda Chaplin , Bradley Davy , Ali Ebrahim , Danny Axford , Takehiko Tosha , Hiroshi Sugimoto , Shigeki Owada , Florian Dworkowski , Ivo Tews , Robin Owen , Michael Hough , Jonathan A. R. Worrall Angewandte Chemie International Edition DOI: 10.1002/anie.202008622 Open Access Show Abstract ▼ Abstract: Obtaining structures of intact redox states of metal centres derived from zero dose X‐ray crystallography can advance our mechanistic understanding of metalloenzymes. In dye‐decolourising 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 (Fe IV =O and a porphyrin cation radical). Using serial femtosecond X‐ray (SFX) crystallography, we have determined the pristine structures of the Fe III and Fe IV =O redox states of a B‐type DyP. These structures reveal a water‐free distal heme site, which together with the presence of an asparagine, infer 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.	Aug 2020
Krios II-Titan Krios II at Diamond	The active form of quinol-dependent nitric oxide reductase from Neisseria meningitidis is a dimer M. Arif M. Jamali , Chai C. Gopalasingam , Rachel M. Johnson , Takehiko Tosha , Kazumasa Muramoto , Stephen P. Muench , Svetlana V. Antonyuk , Yoshitsugu Shiro , Samar Hasnain Iucrj 7, 404 - 415 DOI: 10.1107/S2052252520003656 Diamond Proposal Number(s): [19832] Open Access Show Abstract ▼ Abstract: Neisseria meningitidis 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 N. meningitidis (NmqNOR) 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 NmqNOR, 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 Alcaligenes (Achromobacter) xylosoxidans, 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 NmqNOR to high resolution. This represents one of the first examples in which the two approaches have been used to reveal a monomeric assembly in crystallo 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.	May 2020
Krios II-Titan Krios II at Diamond	Dimeric structures of quinol-dependent nitric oxide reductases (qNORs) revealed by cryo–electron microscopy Chai C. Gopalasingam , Rachel M. Johnson , George N. Chiduza , Takehiko Tosha , Masaki Yamamoto , Yoshitsugu Shiro , Svetlana V. Antonyuk , Stephen P. Muench , S. Samar Hasnain Science Advances 5 DOI: 10.1126/sciadv.aax1803 Diamond Proposal Number(s): [19832] Open Access Show Abstract ▼ Abstract: Quinol-dependent nitric oxide reductases (qNORs) are membrane-integrated, iron-containing enzymes of the denitrification pathway, which catalyze the reduction of nitric oxide (NO) to the major ozone destroying gas nitrous oxide (N2O). Cryo–electron microscopy structures of active qNOR from Alcaligenes xylosoxidans and an activity-enhancing mutant have been determined to be at local resolutions of 3.7 and 3.2 Å, respectively. They unexpectedly reveal a dimeric conformation (also confirmed for qNOR from Neisseria meningitidis) and define the active-site configuration, with a clear water channel from the cytoplasm. Structure-based mutagenesis has identified key residues involved in proton transport and substrate delivery to the active site of qNORs. The proton supply direction differs from cytochrome c–dependent NOR (cNOR), where water molecules from the cytoplasm serve as a proton source similar to those from cytochrome c oxidase.	Aug 2019

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