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Romie C.
Nguyen
,
Ian
Davis
,
Medhanjali
Dasgupta
,
Yifan
Wang
,
Philipp S.
Simon
,
Agata
Butryn
,
Hiroki
Makita
,
Isabel
Bogacz
,
Kednerlin
Dornevil
,
Pierre
Aller
,
Asmit
Bhowmick
,
Ruchira
Chatterjee
,
In-Sik
Kim
,
Tiankun
Zhou
,
Derek
Mendez
,
Daniel W.
Paley
,
Franklin
Fuller
,
Roberto
Alonso Mori
,
Alexander
Batyuk
,
Nicholas K.
Sauter
,
Aaron S.
Brewster
,
Allen M.
Orville
,
Vittal K.
Yachandra
,
Junko
Yano
,
Jan F.
Kern
,
Aimin
Liu
Abstract: The P450 enzyme CYP121 from Mycobacterium tuberculosis catalyzes a carbon–carbon (C–C) bond coupling cyclization of the dityrosine substrate containing a diketopiperazine ring, cyclo(l-tyrosine-l-tyrosine) (cYY). An unusual high-spin (S = 5/2) ferric intermediate maximizes its population in less than 5 ms in the rapid freeze-quenching study of CYP121 during the shunt reaction with peracetic acid or hydrogen peroxide in acetic acid solution. We show that this intermediate can also be observed in the crystalline state by EPR spectroscopy. By developing an on-demand-rapid-mixing method for time-resolved serial femtosecond crystallography with X-ray free-electron laser (tr-SFX-XFEL) technology covering the millisecond time domain and without freezing, we structurally monitored the reaction in situ at room temperature. After a 200 ms peracetic acid reaction with the cocrystallized enzyme–substrate microcrystal slurry, a ferric-hydroperoxo intermediate is observed, and its structure is determined at 1.85 Å resolution. The structure shows a hydroperoxyl ligand between the heme and the native substrate, cYY. The oxygen atoms of the hydroperoxo are 2.5 and 3.2 Å from the iron ion. The end-on binding ligand adopts a near-side-on geometry and is weakly associated with the iron ion, causing the unusual high-spin state. This compound 0 intermediate, spectroscopically and structurally observed during the catalytic shunt pathway, reveals a unique binding mode that deviates from the end-on compound 0 intermediates in other heme enzymes. The hydroperoxyl ligand is only 2.9 Å from the bound cYY, suggesting an active oxidant role of the intermediate for direct substrate oxidation in the nonhydroxylation C–C bond coupling chemistry.
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Nov 2023
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Hugo
Lebrette
,
Vivek
Srinivas
,
Juliane
John
,
Oskar
Aurelius
,
Rohit
Kumar
,
Daniel
Lundin
,
Aaron S.
Brewster
,
Asmit
Bhowmick
,
Abhishek
Sirohiwal
,
In-Sik
Kim
,
Sheraz
Gul
,
Cindy
Pham
,
Kyle D.
Sutherlin
,
Philipp
Simon
,
Agata
Butryn
,
Pierre
Aller
,
Allen M.
Orville
,
Franklin D.
Fuller
,
Roberto
Alonso-Mori
,
Alexander
Batyuk
,
Nicholas K.
Sauter
,
Vittal K.
Yachandra
,
Junko
Yano
,
Ville R. I.
Kaila
,
Britt-Marie
Sjöberg
,
Jan
Kern
,
Katarina
Roos
,
Martin
Högbom
Abstract: Aerobic ribonucleotide reductases (RNRs) initiate synthesis of DNA building blocks by generating a free radical within the R2 subunit; the radical is subsequently shuttled to the catalytic R1 subunit through proton-coupled electron transfer (PCET). We present a high-resolution room temperature structure of the class Ie R2 protein radical captured by x-ray free electron laser serial femtosecond crystallography. The structure reveals conformational reorganization to shield the radical and connect it to the translocation path, with structural changes propagating to the surface where the protein interacts with the catalytic R1 subunit. Restructuring of the hydrogen bond network, including a notably short O–O interaction of 2.41 angstroms, likely tunes and gates the radical during PCET. These structural results help explain radical handling and mobilization in RNR and have general implications for radical transfer in proteins.
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Oct 2023
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Juliane
John
,
Oskar
Aurelius
,
Vivek
Srinivas
,
Patricia
Saura
,
In-Sik
Kim
,
Asmit
Bhowmick
,
Philipp S.
Simon
,
Medhanjali
Dasgupta
,
Cindy
Pham
,
Sheraz
Gul
,
Kyle D.
Sutherlin
,
Pierre
Aller
,
Agata
Butryn
,
Allen M.
Orville
,
Mun Hon
Cheah
,
Shigeki
Owada
,
Kensuke
Tono
,
Franklin D
Fuller
,
Alexander
Batyuk
,
Aaron S.
Brewster
,
Nicholas K.
Sauter
,
Vittal K
Yachandra
,
Junko
Yano
,
Ville R. I.
Kaila
,
Jan
Kern
,
Hugo
Lebrette
,
Martin
Högbom
Open Access
Abstract: Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here, we describe structural rearrangements and crosstalk within the Bacillus cereus ribonucleotide reductase R2b–NrdI complex, a di-metal carboxylate-flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction. Together with density functional theory calculations, we show that the flavin is under steric strain in the R2b–NrdI protein complex, likely tuning its redox properties to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O2 interaction site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b–NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.
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Sep 2022
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I24-Microfocus Macromolecular Crystallography
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Agata
Butryn
,
Philipp S.
Simon
,
Pierre
Aller
,
Philip
Hinchliffe
,
Ramzi N.
Massad
,
Gabriel
Leen
,
Catherine L.
Tooke
,
Isabel
Bogacz
,
In-Sik
Kim
,
Asmit
Bhowmick
,
Aaron S.
Brewster
,
Nicholas E.
Devenish
,
Jurgen
Brem
,
Jos J. A. G.
Kamps
,
Pauline A.
Lang
,
Patrick
Rabe
,
Danny
Axford
,
John H.
Beale
,
Bradley
Davy
,
Ali
Ebrahim
,
Julien
Orlans
,
Selina L. S.
Storm
,
Tiankun
Zhou
,
Shigeki
Owada
,
Rie
Tanaka
,
Kensuke
Tono
,
Gwyndaf
Evans
,
Robin L.
Owen
,
Frances A.
Houle
,
Nicholas K.
Sauter
,
Christopher J.
Schofield
,
James
Spencer
,
Vittal K.
Yachandra
,
Junko
Yano
,
Jan F.
Kern
,
Allen M.
Orville
Diamond Proposal Number(s):
[19458, 25260]
Open Access
Abstract: Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.
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Jul 2021
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Vivek
Srinivas
,
Rahul
Banerjee
,
Hugo
Lebrette
,
Jason C.
Jones
,
Oskar
Aurelius
,
In-Sik
Kim
,
Cindy C.
Pham
,
Sheraz
Gul
,
Kyle
Sutherlin
,
Asmit
Bhowmick
,
Juliane
John
,
Esra
Bozkurt
,
Thomas
Fransson
,
Pierre
Aller
,
Agata
Butryn
,
Isabel
Bogacz
,
Philipp Stefan
Simon
,
Stephen
Keable
,
Alexander
Britz
,
Kensuke
Tono
,
Kyung-Sook
Kim
,
Sang-Youn
Park
,
Sang-Jae
Lee
,
Jaehyun
Park
,
Roberto
Alonso-Mori
,
Franklin
Fuller
,
Alexander
Batyuk
,
Aaron S.
Brewster
,
Uwe
Bergmann
,
Nicholas
Sauter
,
Allen M.
Orville
,
Vittal K.
Yachandra
,
Junko
Yano
,
John D.
Lipscomb
,
Jan F.
Kern
,
Martin
Högbom
Abstract: Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme that catalyzes the conversion of methane to methanol at ambient temperature using a nonheme, oxygen-bridged dinuclear iron cluster in the active site. Structural changes in the hydroxylase component (sMMOH) containing the diiron cluster caused by complex formation with a regulatory component (MMOB) and by iron reduction are important for the regulation of O2 activation and substrate hydroxylation. Structural studies of metalloenzymes using traditional synchrotron-based X-ray crystallography are often complicated by partial X-ray-induced photoreduction of the metal center, thereby obviating determination of the structure of pure oxidation states. Here microcrystals of the sMMOH:MMOB complex from Methylosinus trichosporium OB3b were serially exposed to X-ray free electron laser (XFEL) pulses, where the ≦35 fs duration of exposure of an individual crystal yields diffraction data before photoreduction-induced structural changes can manifest. Merging diffraction patterns obtained from thousands of crystals generates radiation damage free, 1.95 Å resolution crystal structures for the fully oxidized and fully reduced states of the sMMOH:MMOB complex for the first time. The results provide new insight into the manner by which the diiron cluster and the active site environment are reorganized by the regulatory protein component in order to enhance the steps of oxygen activation and methane oxidation. This study also emphasizes the value of XFEL and serial femtosecond crystallography (SFX) methods for investigating the structures of metalloenzymes with radiation sensitive metal active sites.
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Jul 2020
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Mohamed
Ibrahim
,
Thomas
Fransson
,
Ruchira
Chatterjee
,
Mun Hon
Cheah
,
Rana
Hussein
,
Louise
Lassalle
,
Kyle D.
Sutherlin
,
Iris D.
Young
,
Franklin D.
Fuller
,
Sheraz
Gul
,
In-Sik
Kim
,
Philipp S.
Simon
,
Casper
De Lichtenberg
,
Petko
Chernev
,
Isabel
Bogacz
,
Cindy C.
Pham
,
Allen M.
Orville
,
Nicholas
Saichek
,
Trent
Northen
,
Alexander
Batyuk
,
Sergio
Carbajo
,
Roberto
Alonso-Mori
,
Kensuke
Tono
,
Shigeki
Owada
,
Asmit
Bhowmick
,
Robert
Bolotovsky
,
Derek
Mendez
,
Nigel W.
Moriarty
,
James M.
Holton
,
Holger
Dobbek
,
Aaron S.
Brewster
,
Paul D.
Adams
,
Nicholas K.
Sauter
,
Uwe
Bergmann
,
Athina
Zouni
,
Johannes
Messinger
,
Jan
Kern
,
Vittal K.
Yachandra
,
Junko
Yano
Open Access
Abstract: In oxygenic photosynthesis, light-driven oxidation of water to molecular oxygen is carried out by the oxygen-evolving complex (OEC) in photosystem II (PS II). Recently, we reported the room-temperature structures of PS II in the four (semi)stable S-states, S1, S2, S3, and S0, showing that a water molecule is inserted during the S2 → S3 transition, as a new bridging O(H)-ligand between Mn1 and Ca. To understand the sequence of events leading to the formation of this last stable intermediate state before O2 formation, we recorded diffraction and Mn X-ray emission spectroscopy (XES) data at several time points during the S2 → S3 transition. At the electron acceptor site, changes due to the two-electron redox chemistry at the quinones, QA and QB, are observed. At the donor site, tyrosine YZ and His190 H-bonded to it move by 50 µs after the second flash, and Glu189 moves away from Ca. This is followed by Mn1 and Mn4 moving apart, and the insertion of OX(H) at the open coordination site of Mn1. This water, possibly a ligand of Ca, could be supplied via a “water wheel”-like arrangement of five waters next to the OEC that is connected by a large channel to the bulk solvent. XES spectra show that Mn oxidation (τ of ∼350 µs) during the S2 → S3 transition mirrors the appearance of OX electron density. This indicates that the oxidation state change and the insertion of water as a bridging atom between Mn1 and Ca are highly correlated.
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May 2020
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[11904]
Abstract: Ionic liquid (IL) mixtures enable the design of fluids with finely tuned structural and physicochemical properties for myriad applications. In order to rationally develop and design IL mixtures with the desired properties, a thorough understanding of the structural origins of their physicochemical properties and the thermodynamics of mixing needs to be developed. To elucidate the structural origins of the excess molar volume within IL mixtures containing ions with different alkyl chain lengths, 3 IL mixtures containing 1-alkyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ILs have been explored in a joint small angle X-ray scattering (SAXS) and 129Xe NMR study. The apolar domains of the IL mixtures were shown to possess similar dimensions to the largest alkyl chain of the mixture with the size evolution determined by whether the shorter alkyl chain was able to interact with the apolar domain. 129Xe NMR results illustrated that the origin of excess molar volume in these mixtures was due to fluctuations within these apolar domains arising from alkyl chain mismatch, with the formation of a greater number of smaller voids within the IL structure. These results indicate that free volume effects for these types of mixtures can be predicted from simple considerations of IL structure and that the structural basis for the formation of excess molar volume in these mixtures is substantially different to IL mixtures formed of different types of ions.
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Mar 2019
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I22-Small angle scattering & Diffraction
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Nicholas J.
Brooks
,
Franca
Castiglione
,
Cara
Doherty
,
Andrew
Dolan
,
Anita J.
Hill
,
Patricia A.
Hunt
,
Richard P.
Matthews
,
Michele
Mauri
,
Andrea
Mele
,
Roberto
Simonutti
,
Ignacio J.
Villar Garcia
,
Cameron C.
Weber
,
Tom
Welton
Diamond Proposal Number(s):
[11904]
Open Access
Abstract: The formation of ionic liquid (IL) mixtures has been proposed as an approach to rationally fine-tune the physicochemical properties of ILs for a variety of applications. However, the effects of forming such mixtures on the resultant properties of the liquids are only beginning to be understood. Towards a more complete understanding of both the thermodynamics of mixing ILs and the effect of mixing these liquids on their structures and physicochemical properties, the spatial arrangement and free volume of IL mixtures containing the common [C4C1im]+ cation and different anions have been systematically explored using small angle X-ray scattering (SAXS), positron annihilation lifetime spectroscopy (PALS) and 129Xe NMR techniques. Anion size has the greatest effect on the spatial arrangement of the ILs and their mixtures in terms of the size of the non-polar domains and inter-ion distances. It was found that differences in coulombic attraction between oppositely charged ions arising from the distribution of charge density amongst the atoms of the anion also significantly influences these inter-ion distances. PALS and 129Xe NMR results pertaining to the free volume of these mixtures were found to strongly correlate with each other despite the vastly different timescales of these techniques. Furthermore, the excess free volumes calculated from each of these measurements were in excellent agreement with the excess volumes of mixing measured for the IL mixtures investigated. The correspondence of these techniques indicates that the static and dynamic free volume of these liquid mixtures are strongly linked. Consequently, fluxional processes such as hydrogen bonding do not significantly contribute to the free volumes of these liquids compared to the spatial arrangement of ions arising from their size, shape and coulombic attraction. Given the relationship between free volume and transport properties such as viscosity and conductivity, these results provide a link between the structures of IL mixtures, the thermodynamics of mixing and their physicochemical properties.
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Jul 2017
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