I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Hannah R.
Adams
,
Dimitri A.
Svistunenko
,
Michael T.
Wilson
,
Sotaro
Fujii
,
Richard W.
Strange
,
Zoe A.
Hardy
,
Priscilla A.
Vazquez
,
Tyler
Dabritz
,
Gabriel J.
Streblow
,
Colin R.
Andrew
,
Michael A.
Hough
Diamond Proposal Number(s):
[13467, 18565, 25108]
Open Access
Abstract: The structural basis by which gas-binding heme proteins control their interactions with NO, CO, and O2, is fundamental to enzymology, biotechnology and human health. Cytochromes c´ (cyts c´) are a group of putative NO-binding heme proteins that fall into two families: the well characterised four alpha helix bundle fold (cyts c´-α) and an unrelated family with a largely beta sheet fold (cyts c´-β) resembling that of cytochromes P460. A recent structure of cyt c´-β from Methylococcus capsulatus Bath (McCP-β) revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas binding site. This feature, dubbed the “Phe cap”, is highly conserved within the sequences of other cyts c´-β, but is absent in their close homologues, the hydroxylamine oxidizing cytochromes P460, although some do contain a single Phe residue. Here we report an integrated structural, spectroscopic, and kinetic characterization of McCP-β complexes with diatomic gases, focusing on the interaction of the Phe cap with NO and CO. Significantly, crystallographic and resonance Raman data show that orientation of the electron rich aromatic ring face of Phe 32 towards distally-bound NO or CO is associated with weakened backbonding and higher off rates. Moreover, we propose that an aromatic quadrupole also contributes to the unusually weak backbonding reported for some heme-based gas sensors, including the mammalian NO-sensor, soluble guanylate cyclase (sGC). Collectively, this study sheds light on the influence of highly conserved distal Phe residues on heme-gas complexes of cytochrome c’−β, including the potential for aromatic quadrupoles to modulate NO and CO binding in other heme proteins.
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Apr 2023
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18565]
Open Access
Abstract: Structure determination of proteins and enzymes by X-ray crystallography remains the most widely used approach to complement functional and mechanistic studies. Capturing the structures of intact redox states in metalloenzymes is critical for assigning the chemistry carried out by the metal in the catalytic cycle. Unfortunately, X-rays interact with protein crystals to generate solvated photoelectrons that can reduce redox active metals and hence change the coordination geometry and the coupled protein structure. Approaches to mitigate such site-specific radiation damage continue to be developed, but nevertheless application of such approaches to metalloenzymes in combination with mechanistic studies are often overlooked. In this review, we summarize our recent structural and kinetic studies on a set of three heme peroxidases found in the bacterium Streptomyces lividans that each belong to the dye decolourizing peroxidase (DyP) superfamily. Kinetically, each of these DyPs has a distinct reactivity with hydrogen peroxide. Through a combination of low dose synchrotron X-ray crystallography and zero dose serial femtosecond X-ray crystallography using an X-ray free electron laser (XFEL), high-resolution structures with unambiguous redox state assignment of the ferric and ferryl (FeIV = O) heme species have been obtained. Experiments using stopped-flow kinetics, solvent-isotope exchange and site-directed mutagenesis with this set of redox state validated DyP structures have provided the first comprehensive kinetic and structural framework for how DyPs can modulate their distal heme pocket Asp/Arg dyad to use either the Asp or the Arg to facilitate proton transfer and rate enhancement of peroxide heterolysis.
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Sep 2021
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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
Open Access
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.
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Aug 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18565]
Open Access
Abstract: Ferritins are multimers comprised of 4 α-helical bundle monomers that co-assemble to form protein shells surrounding an approximately spherical internal cavity. The assembled multimers acquire Fe2+ from their surroundings by utilising channels that penetrate the protein for the transportation of iron to diiron catalytic centres buried within the monomeric units. Here oxidation of the substrate to Fe3+ is coupled to the reduction of O2 and/or peroxide to yield the precursor to a ferric oxy hydroxide mineral that is stored within the internal cavity. The rhombic dodecahedral quaternary structure results in channels of 4-fold and 3-fold symmetry, located at the vertices, which are common to all 24mer-ferritins. Ferritins isolated from higher eukaryotes have been demonstrated to take up Fe2+ via the 3-fold channels. One of the defining features of ferritins isolated from prokaryotes is the presence of a further 24 channels, the B-channels, and these are thought to play an important role in Fe2+ uptake in this sub-family. SynFtn is an unusual ferritin isolated from the marine cyanobacterium Synechococcus CC9311. The reported structure of SynFtn derived from Fe2+ soaked crystals revealed the presence of a fully hydrated Fe2+ associated with three aspartate residues (Asp137 from each of the three symmetry related subunits) within each three-fold channel, suggesting that it might be the route for Fe2+ entry. Here, we present structural and spectro-kinetic data on two variants of SynFtn, D137A and E62A, designed to assess this possibility. Glu62 is equivalent to residues demonstrated to be important in the transfer of iron from the inner exit of the 3-fold channel to the catalytic centre in animal ferritins. As expected replacing Asp137 with a non-coordinating residue eliminated rapid iron oxidation by SynFtn. In contrast the rate of mineral core formation was severely impaired whilst the rate of iron transit into the catalytic centre was largely unaffected upon introducing a non-coordinating residue in place of Glu62 suggesting a role for this residue in release of the oxidised product. The identification of these two residues in SynFtn maps out major routes for Fe2+ entry to, and exit from, the catalytic ferroxidase centres.
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Jan 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9475]
Abstract: The gene encoding the cyanobacterial ferritin SynFtn is up-regulated in response to copper stress. Here, we show that, while SynFtn does not interact directly with copper, it is highly unusual in several ways. First, its catalytic diiron ferroxidase center is unlike those of all other characterized prokaryotic ferritins and instead resembles an animal H-chain ferritin center. Second, as demonstrated by kinetic, spectroscopic, and high-resolution X-ray crystallographic data, reaction of O2 with the di-Fe2+ center results in a direct, one-electron oxidation to a mixed-valent Fe2+/Fe3+ form. Iron–O2 chemistry of this type is currently unknown among the growing family of proteins that bind a diiron site within a four α-helical bundle in general and ferritins in particular. The mixed-valent form, which slowly oxidized to the more usual di-Fe3+ form, is an intermediate that is continually generated during mineralization. Peroxide, rather than superoxide, is shown to be the product of O2 reduction, implying that ferroxidase centers function in pairs via long-range electron transfer through the protein resulting in reduction of O2 bound at only one of the centers. We show that electron transfer is mediated by the transient formation of a radical on Tyr40, which lies ∼4 Å from the diiron center. As well as demonstrating an expansion of the iron–O2 chemistry known to occur in nature, these data are also highly relevant to the question of whether all ferritins mineralize iron via a common mechanism, providing unequivocal proof that they do not.
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Jan 2019
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[7461]
Open Access
Abstract: Nature is adept at utilising highly similar protein folds to carry out very different functions, yet the mechanisms by which this functional divergence occurs remain poorly characterised. In certain methanotrophic bacteria, two homologous pentacoordinate c-type heme proteins have been identified: a cytochrome P460 (cyt P460) and a cytochrome c′-β (cyt cp-β). Cytochromes P460 are able to convert hydroxylamine to nitrous oxide (N2O), a potent greenhouse gas. This reactivity is similar to that of hydroxylamine oxidoreductase (HAO), which is a key enzyme in nitrifying and methanotrophic bacteria. Cyt P460 and HAO both have unusual protein-heme cross-links, formed by a Tyr residue in HAO and a Lys in cyt P460. In contrast, cyts cp-β (the only known cytochromes c′ with a β-sheet fold) lack this crosslink and appears to be optimized for binding non-polar molecules (including NO and CO) without enzymatic conversion. Our bioinformatics analysis supports the proposal that cyt cp-β may have evolved from cyt P460 via a gene duplication event. Using high-resolution X-ray crystallography, UV-visible absorption, electron paramagnetic resonance (EPR) and resonance Raman spectroscopy, we have characterized the overall protein folding and active site structures of cyt cp-β and cyt P460 from the obligate methanotroph, Methylococcus capsulatus (Bath). These proteins display a similar β-sheet protein fold, together with a pattern of changes to the heme pocket regions and localised tertiary structure that have converted a hydroxylamine oxidizing enzyme into a gas-binding protein. Structural comparisons provide insights relevant to enzyme redesign for synthetic enzymology and engineering of gas sensor proteins. We also show the widespread occurrence of cyts cp-β and characterise their phylogeny.
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Jan 2019
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7461]
Abstract: GlxA from Streptomyces lividans is a mononuclear copper-radical oxidase and a member of the auxiliary activity family 5 (AA5). Its domain organisation and low sequence homology make it a distinct member of the AA5 family in which the fungal galactose 6-oxidase (Gox) is the best-characterized. GlxA is a key cuproenzyme in the copper-dependent morphological development of S. lividans with a function that is linked to the processing of an extracytoplasmic glycan. The catalytic site in GlxA and Gox contain two distinct one-electron acceptors comprising the copper ion and a 3'-(S-cysteinyl) tyrosine. The latter is formed post-translationally through a covalent bond between a cysteine and a copper coordinating tyrosine ligand and houses a radical. In GlxA and Gox a second coordination sphere tryptophan residue (Trp288 in GlxA) is present, but the orientation of the indole ring differs between the two enzymes creating a marked difference in the π-π stacking interaction of the benzyl ring with the 3'-(S-cysteinyl) tyrosine. Differences in the spectroscopic and enzymatic activity have been reported between GlxA and Gox with the indole orientation suggested as a reason. Here we report a series of in vivo and in vitro studies using the W288F and W288A variants of GlxA to assess the role of Trp288 on the morphology, maturation, spectroscopic and enzymatic properties. Our findings point towards a salient role for Trp288 in the kinetics of copper loading and maturation of GlxA, with its presence essential for stabilising the metalloradical site required for coupling catalytic activity and morphological development.
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Jan 2017
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7461]
Abstract: Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively deconstruct polysaccharides. The active site copper in LPMOs is coordinated by a histidine-brace. This utilizes the amino group and side chain of the N-terminal His residue with the side chain of a second His residue to create a T-shaped arrangement of nitrogen ligands. We report a structural, kinetic, and thermodynamic appraisal of copper binding to the histidine-brace in an auxiliary activity family 10 (AA10) LPMO from Streptomyces lividans (SliLPMO10E). Unexpectedly, we discovered the existence of two apo-SliLPMO10E species in solution that can each bind copper at a single site with distinct kinetic and thermodynamic (exothermic and endothermic) properties. The experimental EPR spectrum of copper-bound SliLPMO10E requires the simulation of two different line shapes, implying two different copper-bound species, indicative of three and two nitrogen ligands coordinating the copper. Amino group coordination was probed through the creation of an N-terminal extension variant (SliLPMO10E-Ext). The kinetics and thermodynamics of copper binding to SliLPMO10E-Ext are in accord with copper binding to one of the apo-forms in the wild-type protein, suggesting that amino group coordination is absent in the two-nitrogen coordinate form of SliLPMO10E. Copper binding to SliLPMO10B was also investigated, and again it revealed the presence of two apo-forms with kinetics and stoichiometry of copper binding identical to that of SliLPMO10E. Our findings highlight that heterogeneity exists in the active site copper coordination sphere of LPMOs that may have implications for the mechanism of loading copper in the cell.
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Jun 2016
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I03-Macromolecular Crystallography
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Chukwudi I.
Nnamchi
,
Gary
Parkin
,
Igor
Efimov
,
Jaswir
Basran
,
Hanna
Kwon
,
Dimitri A.
Svistunenko
,
Jon
Agirre
,
Bartholomew N.
Okolo
,
Anene
Moneke
,
Bennett C.
Nwanguma
,
Peter
Moody
,
Emma L.
Raven
Diamond Proposal Number(s):
[6388]
Open Access
Abstract: A cationic class III peroxidase from Sorghum
bicolor was purified to homogeneity. The enzyme contains
a high-spin heme, as evidenced by UV–visible spectroscopy
and EPR. Steady state oxidation of guaiacol was
demonstrated and the enzyme was shown to have higher
activity in the presence of calcium ions. A FeIII/FeII reduction
potential of −266 mV vs NHE was determined.
Stopped-flow experiments with H2O2 showed formation
of a typical peroxidase Compound I species, which converts
to Compound II in the presence of calcium. A crystal
structure of the enzyme is reported, the first for a sorghum
peroxidase. The structure reveals an active site that
is analogous to those for other class I heme peroxidase, and
a substrate binding site (assigned as arising from binding of indole-3-acetic acid) at the γ-heme edge. Metal binding
sites are observed in the structure on the distal (assigned
as a Na+ ion) and proximal (assigned as a Ca2+) sides of
the heme, which is consistent with the Ca2+-dependence of
the steady state and pre-steady state kinetics. It is probably
the case that the structural integrity (and, thus, the catalytic
activity) of the sorghum enzyme is dependent on metal ion
incorporation at these positions.
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Dec 2015
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9475]
Open Access
Abstract: Ferritins are iron storage proteins that overcome the problems of toxicity and poor bioavailability of iron by catalyzing iron oxidation and mineralization through the activity of a diiron ferroxidase site. Unlike in other ferritins, the oxidized di-Fe3+ site of Escherichia coli bacterioferritin (EcBFR) is stable and therefore does not function as a conduit for the transfer of Fe3+ into the storage cavity, but instead acts as a true catalytic cofactor that cycles its oxidation state while driving Fe2+ oxidation in the cavity. Herein, we demonstrate that EcBFR mineralization depends on three aromatic residues near the diiron site, Tyr25, Tyr58, and Trp133, and that a transient radical is formed on Tyr25. The data indicate that the aromatic residues, together with a previously identified inner surface iron site, promote mineralization by ensuring the simultaneous delivery of two electrons, derived from Fe2+ oxidation in the BFR cavity, to the di-ferric catalytic site for safe reduction of O2.
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Oct 2015
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