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Abstract: X-ray induced photoreduction of macromolecular structures has been well reported, with the accompanying site-specific radiation damage occurring in a predictable order. Metal containing centres are reduced first, followed by disulphide reduction, decarboxylation of glutamate and aspartate residues and then increased side chain mobility [1]. Photoreduction of the bulk solvent also occurs which contributes to global radiation damage, identifiable in data processing statistics. In a recent study into the structure of a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from a human pathogen, the active site cysteine was noted to be modified to a sulfininc acid (R-SO(OH)). GAPDH is a core metabolic enzyme, involved in ATP and pyruvate generation by catalysing the reversible oxidative phosphorylation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate [2]. An oxidised active site cysteine is incompatible with the current reaction mechanism for GAPDHs [3]. This type of modification has been noted previously for GAPDHs and has been attributed to post-translational modifications by reactive oxygen species [4, 5]. The same modification, however, has been noted for other oxidoreductases and postulated to be a form of site-specific radiation damage, where the activated cysteine is oxidised by hydroxyl radicals formed in the bulk solvent [1, 6]. In this work we have mined the PDB for all X-ray structures of GAPDHs and devised a workflow to identify damaged cysteines. Of the 225 structures, 68 were identified as damaged. The damage appears to be decoupled from conventional metrics for identifying specific radiation damage (e.g., Bnet-percentile [7]) and is independent of data collection source or temperature. The method implemented is highly sensitive to damaged cysteines and is effective in screening large datasets. This work will be expanded to search the whole PDB and correlate with other thiol-active site enzymes. Oxidised cysteines being incorrectly modelled as the reduced thiol, whilst likely physiologically accurate, means the built model would have a different electrostatic and steric environment which does not match the electron density. This has a compounding effect on any errors with deep learning and other modelling tools for accurate model building or automated drug discovery pipelines, which use this data for their training.
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Jun 2025
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[31440]
Open Access
Abstract: Horseradish peroxidase (HRP), isolated from horseradish roots, is heavily glycosylated, making it difficult to crystallize. In this work, we produced recombinant HRP in E. coli and obtained an X-ray structure of the ferric enzyme at 1.63 Å resolution. The structure shows that the recombinant HRP contains four disulphide bonds and two calcium ions, which are highly conserved in class III peroxidase enzymes. The heme active site contains histidine residues at the proximal (His 170) and distal (His 42) positions, and an active site arginine (Arg 38). Surprisingly, an ethylene glycol molecule was identified in the active site, forming hydrogen bonds with His 42 and Arg 38 at the δ-heme edge. The high yields obtained from the recombinant expression system, and the successful crystallization of the enzyme pave the way for new structural studies in the future.
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Mar 2025
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I03-Macromolecular Crystallography
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Samuel L.
Freeman
,
Vera
Skafar
,
Hanna
Kwon
,
Alistair J.
Fielding
,
Peter C. E.
Moody
,
Alejandra
Martínez
,
Federico
Issoglio
,
Lucas
Inchausti
,
Pablo
Smircich
,
Ari
Zeida
,
Lucía
Piacenza
,
Rafael
Radi
,
Emma L.
Raven
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: The protozoan parasite Trypanosoma cruzi is the causative agent of American trypanosomiasis, otherwise known as Chagas disease. To survive in the host, the T. cruzi parasite needs antioxidant defence systems. One of these is a hybrid heme peroxidase, the T. cruzi ascorbate peroxidase-cytochrome c peroxidase enzyme (TcAPx-CcP). TcAPx-CcP has high sequence identity to members of the class I peroxidase family, notably ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP), as well as a mitochondrial peroxidase from Leishmania major (LmP). The aim of this work was to solve the structure and examine the reactivity of the TcAPx-CcP enzyme. Low temperature electron paramagnetic resonance (EPR) spectra support the formation of an exchange-coupled [Fe(IV)=O Trp233•+] Compound I radical species, analogous to that used in CcP and LmP. We demonstrate that TcAPx-CcP is similar in overall structure to APX and CcP, but there are differences in the substrate binding regions. Furthermore, the electron transfer pathway from cytochrome c to the heme in CcP and LmP is preserved in the TcAPx-CcP structure. Integration of steady state kinetic experiments, molecular dynamic simulations, and bioinformatic analyses indicates that TcAPx-CcP preferentially oxidizes cytochrome c, but is still competent for oxididation of ascorbate. The results reveal that TcAPx-CcP is a credible cytochrome c peroxidase which can also bind and use ascorbate in host cells, where concentrations are in the millimolar range. Thus, kinetically and functionally TcAPx-CcP can be considered a hybrid peroxidase.
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Jun 2022
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I03-Macromolecular Crystallography
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Abstract: Campylobacter jejuni is a pathogenic bacteria that causes gastrointestinal disorders and is thus of great importance. Phosphorylating Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a ubiquitous cellular enzyme that has a well-defined role in glycolysis and other pathways where it catalyses the oxidative phosphorylation of glyceraldehyde 3-phosphate (2-hydroxy-3-oxopropyl dihydrogen phosphate) to 1,3-Bisphosphoglycerate ((2-Hydroxy-3-phosphonooxy-propanoyloxy)phosphonic acid). The C. jejuni genome encodes a single GAPDH enzyme (CjGAPDH) which displays dual (NAD/NADP) coenzyme specificity. NAD-specific GAPDHs are given the EC classification of 1.2.1.12, whereas NADP-specific GAPDHs are classed as 1.2.1.13. GAPDH's with dual specificity are in the class 1.2.1.59. Here we present the X-ray crystal structure of this enzyme (at 2.25 Å), this comprises superimposed structures of NAD- and NADP- complexes showing the structural adaptation that allows this dual specificity, and we consider this in the context of the pathogen's metabolism. There are no previous reports of EC 1.2.1.59 structures that compare the binding of the two co-enzymes. Furthermore, we also report the structure (at 2.05 Å) of the enzyme complexed with the nucleoside ADP and consider this with respect to the reported “moonlighting” activities of GAPDH.
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Jun 2021
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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
Open Access
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.
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Apr 2021
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I03-Macromolecular Crystallography
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Mark J
Burton
,
Joel
Cresser-Brown
,
Morgan
Thomas
,
Nicola
Portolano
,
Jaswir
Basran
,
Samuel L.
Freeman
,
Hanna
Kwon
,
Andrew R.
Bottrill
,
Manuel J
Llansola-Portoles
,
Andrew A
Pascal
,
Rebekah
Jukes-Jones
,
Tatyana
Chernova
,
Ralf
Schmid
,
Noel W.
Davies
,
Nina M.
Storey
,
Pierre
Dorlet
,
Peter C. E.
Moody
,
John S
Mitcheson
,
Emma L.
Raven
Diamond Proposal Number(s):
[14692]
Open Access
Abstract: The ether-à-go-go (EAG) family of voltage gated K+ channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer and sudden cardiac death. A defining feature of EAG (Kv10-12) channels is a highly conserved domain on the amino-terminus, known as the eag-domain, consisting of a PAS domain capped by a short sequence containing an amphipathic helix (Cap-domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pull-down assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in E.coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells.
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Jul 2020
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[14692, 10369, 8359]
Open Access
Abstract: The multiprotein complex C1 initiates the classical pathway of complement activation on binding to antibody–antigen complexes, pathogen surfaces, apoptotic cells, and polyanionic structures. It is formed from the recognition subcomponent C1q and a tetramer of proteases C1r2C1s2 as a Ca2+-dependent complex. Here we have determined the structure of a complex between the CUB1-EGF-CUB2 fragments of C1r and C1s to reveal the C1r–C1s interaction that forms the core of C1. Both fragments are L-shaped and interlock to form a compact antiparallel heterodimer with a Ca2+ from each subcomponent at the interface. Contacts, involving all three domains of each protease, are more extensive than those of C1r or C1s homodimers, explaining why heterocomplexes form preferentially. The available structural and biophysical data support a model of C1r2C1s2 in which two C1r-C1s dimers are linked via the catalytic domains of C1r. They are incompatible with a recent model in which the N-terminal domains of C1r and C1s form a fixed tetramer. On binding to C1q, the proteases become more compact, with the C1r-C1s dimers at the center and the six collagenous stems of C1q arranged around the perimeter. Activation is likely driven by separation of the C1r-C1s dimer pairs when C1q binds to a surface. Considerable flexibility in C1s likely facilitates C1 complex formation, activation of C1s by C1r, and binding and activation of downstream substrates C4 and C4b-bound C2 to initiate the reaction cascade.
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Jan 2018
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692, 10369, 8359, 6388, 310]
Abstract: Aerobic organisms have evolved to activate oxygen from the atmosphere, which allows them to catalyze the oxidation of different kinds of substrates. This activation of oxygen is achieved by a metal center (usually iron or copper) buried within a metalloprotein. In the case of iron-containing heme enzymes, the activation of oxygen is achieved by formation of transient iron-oxo (ferryl) intermediates; these intermediates are called Compound I and Compound II. The Compound I and II intermediates were first discovered in the 1930s in horseradish peroxidase, and it is now known that these same species are used across the family of heme enzymes, which include all of the peroxidases, the heme catalases, the P450s, cytochrome c oxidase, and NO synthase. Many years have passed since the first observations, but establishing the chemical nature of these transient ferryl species remains a fundamental question that is relevant to the reactivity, and therefore the usefulness, of these species in biology.
This Account summarizes experiments that were conceived and conducted at Leicester and presents our ideas on the chemical nature, stability, and reactivity of these ferryl heme species. We begin by briefly summarizing the early milestones in the field, from the 1940s and 1950s. We present comparisons between the nature and reactivity of the ferryl species in horseradish peroxidase, cytochrome c peroxidase, and ascorbate peroxidase; and we consider different modes of electron delivery to ferryl heme, from different substrates in different peroxidases.
We address the question of whether the ferryl heme is best formulated as an (unprotonated) FeIV═O or as a (protonated) FeIV–OH species. A range of spectroscopic approaches (EXAFS, resonance Raman, Mossbauer, and EPR) have been used over many decades to examine this question, and in the last ten years, X-ray crystallography has also been employed. We describe how information from all of these studies has blended together to create an overall picture, and how the recent application of neutron crystallography has directly identified protonation states and has helped to clarify the precise nature of the ferryl heme in cytochrome c peroxidase and ascorbate peroxidase. We draw comparisons between the Compound I and Compound II species that we have observed in peroxidases with those found in other heme systems, notably the P450s, highlighting possible commonality across these heme ferryl systems. The identification of proton locations from neutron structures of these ferryl species opens the door for understanding the proton translocations that need to occur during O–O bond cleavage.
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Jan 2018
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I04-Macromolecular Crystallography
|
Hanna
Kwon
,
Jaswir
Basran
,
Cecilia M.
Casadei
,
Alistair J.
Fielding
,
Tobias E.
Schrader
,
Andreas
Ostermann
,
Juliette M.
Devos
,
Pierre
Aller
,
Matthew P.
Blakeley
,
P. C. E.
Moody
,
Emma L.
Raven
Diamond Proposal Number(s):
[10369]
Open Access
Abstract: Catalytic heme enzymes carry out a wide range of oxidations in biology. They have in common a mechanism that requires formation of highly oxidized ferryl intermediates. It is these ferryl intermediates that provide the catalytic engine to drive the biological activity. Unravelling the nature of the ferryl species is of fundamental and widespread importance. The essential question is whether the ferryl is best described as a Fe(IV)=O or a Fe(IV)–OH species, but previous spectroscopic and X-ray crystallographic studies have not been able to unambiguously differentiate between the two species. Here we use a different approach. We report a neutron crystal structure of the ferryl intermediate in Compound II of a heme peroxidase; the structure allows the protonation states of the ferryl heme to be directly observed. This, together with pre-steady state kinetic analyses, electron paramagnetic resonance spectroscopy and single crystal X-ray fluorescence, identifies a Fe(IV)–OH species as the reactive intermediate. The structure establishes a precedent for the formation of Fe(IV)–OH in a peroxidase.
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Nov 2016
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Umakhanth
Venkatraman Girija
,
Christopher
Furze
,
Alex
Gingras
,
Takayuki
Yoshizaki
,
Katsuki
Ohtani
,
Jamie E
Marshall
,
A Katrine
Wallis
,
Wilhelm J
Schwaeble
,
Mohammed
El-Mezgueldi
,
Daniel A
Mitchell
,
Peter
Moody
,
Nobutaka
Wakamiya
,
Russell
Wallis
Diamond Proposal Number(s):
[8359]
Open Access
Abstract: Collectin-K1 (CL-K1, or CL-11) is a multifunctional Ca2+-dependent lectin with roles in innate immunity, apoptosis and embryogenesis. It binds to carbohydrates on pathogens to activate the lectin pathway of complement and together with its associated serine protease MASP-3 serves as a guidance cue for neural crest development. High serum levels are associated with disseminated intravascular coagulation, where spontaneous clotting can lead to multiple organ failure. Autosomal mutations in the CL-K1 or MASP-3 genes cause a developmental disorder called 3MC (Carnevale, Mingarelli, Malpuech and Michels) syndrome, characterised by facial, genital, renal and limb abnormalities. One of these mutations (Gly204Ser in the CL-K1 gene) is associated with undetectable levels of protein in the serum of affected individuals.
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Dec 2015
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