Crystal structure of a zinc-containing glycerol dehydrogenase (TM0423) from Thermotoga maritima at 1.5 A resolution.
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Binding Sites↗
Biomedical subjects
Keith O Hodgson
Publications and source records attributed to Keith O Hodgson.
The vanadium environment in blood cells of Ascidia ceratodes is divergent at all organismal levels: an XAS and EPR spectroscopic study.
K-edge X-ray absorption and EPR spectroscopies were used to test the variation in blood cell vanadium between and within specimens of the tunicate Ascidia ceratodes from Bodega Bay, California. Intracellular vanadium was speciated by fitting the XAS spectra of whole blood cells with linear combinations of the XAS spectra of models. Blood cell samples representing one specimen each, respectively, revealed 92.5 and 38.7% of endogenous vanadium as [V(H(2)O)(6)](3+), indicating dissimilar distributions. Conversely, vanadium distributions within blood cell samples respectively representing one and six specimens proved very similar. The derived array of V(III) complexes was consistent with multiple intracellular regions that differ both in pH and c(sulfate), both within and between specimens. No systematic effect on vanadium distribution was apparent on mixing blood cells. EPR and XAS results indicated at least three forms of endogenous vanadyl ion, two of which may be dimeric. An inverse linear correlation was found between soluble and complexed forms of vanadyl ion, implying co-regulation. The EPR A value of endogenous vanadyl ion [A(0)=(1.062+/-0.008)x10(-2) cm(-1)] was marginally different from that representing Monterey Bay A. ceratodes [A(0)=(1.092+/-0.006) x10(-2) cm(-1)]. Comparisons indicate that Bodega Bay A. ceratodes maintain V(III) in a more acidic intracellular environment on average than do those from Monterey Bay, showing variation across populations. Blood cell vanadium thus noticeably diverges at all organismal levels among A. ceratodes.
Animals↗
Imaging whole Escherichia coli bacteria by using single-particle x-ray diffraction.
We report the first experimental recording, to our knowledge, of the diffraction pattern from intact Escherichia coli bacteria using coherent x-rays with a wavelength of 2 A. By using the oversampling phasing method, a real space image at a resolution of 30 nm was directly reconstructed from the diffraction pattern. An R factor used for characterizing the quality of the reconstruction was in the range of 5%, which demonstrated the reliability of the reconstruction process. The distribution of proteins inside the bacteria labeled with manganese oxide has been identified and this distribution confirmed by fluorescence microscopy images. Compared with lens-based microscopy, this diffraction-based imaging approach can examine thicker samples, such as whole cultured cells, in three dimensions with resolution limited only by radiation damage. Looking forward, the successful recording and reconstruction of diffraction patterns from biological samples reported here represent an important step toward the potential of imaging single biomolecules at near-atomic resolution by combining single-particle diffraction with x-ray free electron lasers.
Escherichia coli↗
Crystal structure of thy1, a thymidylate synthase complementing protein from Thermotoga maritima at 2.25 A resolution.
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Crystallography, X-Ray↗
Atomic resolution three-dimensional electron diffraction microscopy.
We report the development of a novel form of diffraction-based 3D microscopy to overcome resolution barriers inherent in high-resolution electron microscopy and tomography. By combining coherent electron diffraction with the oversampling phasing method, we show that the 3D structure of a nanocrystal can be determined ab initio at a resolution of 1 A from 29 simulated noisy diffraction patterns. This new form of microscopy can be used to image the 3D structures of nanocrystals and noncrystalline samples, with resolution limited only by the quality of sample diffraction.
Journal Article↗
Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline.
Structural genomics is emerging as a principal approach to define protein structure-function relationships. To apply this approach on a genomic scale, novel methods and technologies must be developed to determine large numbers of structures. We describe the design and implementation of a high-throughput structural genomics pipeline and its application to the proteome of the thermophilic bacterium Thermotoga maritima. By using this pipeline, we successfully cloned and attempted expression of 1,376 of the predicted 1,877 genes (73%) and have identified crystallization conditions for 432 proteins, comprising 23% of the T. maritima proteome. Representative structures from TM0423 glycerol dehydrogenase and TM0449 thymidylate synthase-complementing protein are presented as examples of final outputs from the pipeline.
Cloning, Molecular↗
Aryl CbondH activation by CuII to form an organometallic aryl-CuIII species: a novel twist on copper disproportionation.
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Copper↗
A stabilized mu-eta(2):eta(2) peroxodicopper(II) complex with a secondary diamine ligand and its tyrosinase-like reactivity.
The activation of dioxygen (O(2)) by Cu(I) complexes is an ubiquitous process in biology and industrial applications. In tyrosinase, a binuclear copper enzyme, a mu-eta(2):eta(2)-peroxodicopper(II) species is generally accepted to be the active oxidant. Reported here is the characterization and reactivity of a stable mu-eta(2):eta(2)-peroxodicopper(II) complex at -80 degrees C using a secondary diamine ligand, N,N'-di-tert-butyl-ethylenediamine (DBED). The spectroscopic characteristics of this complex (UV-vis, resonance Raman) prove to be strongly dependent on the counteranion employed and not on the solvent, suggesting an intimate interaction of the counteranions with the Cu-O(2) cores. This interaction is also supported by solution EXAFS data. This new complex exhibits hydroxylation reactivity by converting phenolates to catechols, proving to be a functional model of tyrosinase. Additional interest in this Cu/O(2) species results from the use of Cu(I)-DBED as a polymerization catalyst of phenols to polyphenylene oxide (PPO) with O(2) as the terminal oxidant.
Copper↗
High resolution 3D x-ray diffraction microscopy.
We have imaged a 2D buried Ni nanostructure at 8 nm resolution using coherent x-ray diffraction and the oversampling phasing method. By employing a 3D imaging reconstruction algorithm, for the first time we have experimentally determined the 3D structure of a noncrystalline nanostructured material at 50 nm resolution. The 2D and 3D imaging resolution is currently limited by the exposure time and the computing power, while the ultimate resolution is limited by the x-ray wavelengths. We believe these results pave the way for the development of atomic resolution 3D x-ray diffraction microscopy.
Microscopy↗
Structural and spectroscopic studies of valence-delocalized diiron(II,III) complexes dupported by carboxylate-only bridging ligands.
The synthesis, molecular structures, and spectroscopic properties of a series of valence-delocalized diiron(II,III) complexes are described. One-electron oxidation of diiron(II) tetracarboxylate complexes afforded the compounds [Fe(2)(mu-O(2)CAr(Tol))(4)L(2)]X, where L = 4-(t)BuC(5)H(4)N (1b), C(5)H(5)N (2b), and THF (3b); X = PF(6)(-) (1b and 3b) and OTf(-) (2b). In 1b-3b, four mu-1,3 carboxylate ligands span relatively short Fe...Fe distances of 2.6633(11)-2.713(3) A. Intense (epsilon = 2700-3200 M(-1) cm(-1)) intervalence charge transfer bands were observed at 620-670 nm. EPR spectroscopy confirmed the S = (9)/(2) ground spin state of 1b-3b, the valence-delocalized nature of which was probed by X-ray absorption spectroscopy. The electron delocalization between paramagnetic metal centers is described by double exchange, which, for the first time, is observed in diiron clusters having no single-atom bridging ligand(s).
Algorithms↗
Cooperative ligation, back-bonding, and possible pyridine-pyridine interactions in tetrapyridine-vanadium(II): a visible and X-ray spectroscopic study.
The binding of pyridine by V(II) in aqueous solution shows evidence for the late onset of cooperativity. The K(1) governing formation of [V(py)](2+) (lambda(max) = 404 nm, epsilon(max) = 1.43 +/- 0.3 M(-1) cm(-1)) was determined spectrophotometrically to be 11.0 +/- 0.3 M(-)(1), while K(1) for isonicotinamide was found to be 5.0 +/- 0.1 M(-1). These values are in the low range for 3d M(2+) ions and indicate that V(II).py back-bonding is not significant in the formation of the 1:1 complex. Titration of 10.5 mM V(II) with pyridine in aqueous solution showed an absorption plateau at about 1 M added pyridine, indicating a reaction terminus. Vanadium K-edge EXAFS analysis of 63 mM V(II) in 2 M pyridine solution revealed six first-shell N/O ligands at 2.14 A and 4 +/- 1 pyridine ligands per V(II). UV/vis absorption spectroscopy indicated that the same terminal V(II) species was present in both experiments. Model calculations showed that in the absence of back-bonding only 2.0 +/- 0.2 and 2.4 +/- 0.2 pyridine ligands would be present, respectively. Cooperativity in multistage binding of pyridine by [V(aq)](2+) is thus indicated. XAS K-edge spectroscopy of crystalline [V(O(3)SCF(3))(2)(py)(4)] and of V(II) in 2 M pyridine solution each exhibited the analogous 1s --> (5)E(g) and 1s --> (5)T(2g) transitions, at 5465.5 and 5467.5 eV, and 5465.2 and 5467.4 eV, respectively, consistent with the EXAFS analysis. In contrast, [V(py)(6)](PF(6))(2) and [V(H(2)O)(6)]SO(4) show four 1s --> 3d XAS transitions suggestive of a Jahn-Teller distorted excited state. Comparison of the M(II)[bond]N(py) bond lengths in V(II) and Fe(II) tetrapyridines shows that the V(II)[bond]N(py) distances are about 0.06 A shorter than predicted from ionic radii. For [VX(2)(R-py)(4)] (X = Cl(-), CF(3)SO(3)(-); R = 4-Et, H, 3-EtOOC), the E(1/2) values of the V(II)/V(III) couples correlate linearly with the Hammett sigma values of the R group. These findings indicate that pi back-bonding is important in [V(py)(4)](2+) even though absent in [V(py)](2+). The paramagnetism of [V(O(3)SCF(3))(2)(py)(4)] in CHCl(3), 3.8 +/- 0.2 mu(B), revealed that the onset of back-bonding is not accompanied by a spin change. Analysis of the geometries of V(II) and Fe(II) tetrapyridines indicates that the ubiquitous propeller motif accompanying tetrapyridine ligation may be due to eight dipole interactions arising from the juxtaposed C-H edges and pi clouds of adjoining ligands, worth about -6 kJ each. However, this is not the source of the cooperativity in the binding of multiple pyridines by V(II) because the same interactions are present in the Fe(II)-tetrapyridines, which do not show cooperative ligand binding. Cooperativity in the binding of pyridine by V(II) is then assigned by default to V(II)-pyridine back-bonding, which emerges only after the first pyridine is bound.
Crystallography, X-Ray↗
Nature of the intermediate formed in the reduction of O(2) to H(2)O at the trinuclear copper cluster active site in native laccase.
The multicopper oxidases contain at least four copper atoms and catalyze the four-electron reduction of O(2) to H(2)O at a trinuclear copper cluster. An intermediate, termed native intermediate, has been trapped by a rapid freeze-quench technique from Rhus vernicifera laccase when the fully reduced form reacts with dioxygen. This intermediate had been described as an oxygen-radical bound to the trinuclear copper cluster with one Cu site reduced. XAS, however, shows that all copper atoms are oxidized in this intermediate. A combination of EXAFS, multifrequency EPR, and VTVH MCD has been used to understand how this fully oxidized trinuclear Cu cluster relates to the fully oxidized resting form of the enzyme. It is determined that in the native intermediate all copper atoms of the cluster are bridged by the product of full O(2) reduction. In contrast, the resting form has one copper atom of the cluster (the T2 Cu) magnetically isolated from the others. The native intermediate decays to the resting oxidized form with a rate that is too slow to be in the catalytic cycle. Thus, the native intermediate appears to be the catalytically relevant fully oxidized form of the enzyme, and its role in catalysis is considered.
Binding Sites↗
X-ray absorption spectroscopic investigation of the resting ferrous and cosubstrate-bound active sites of phenylalanine hydroxylase.
Previous studies of ferrous wild-type phenylalanine hydroxylase, [Fe(2+)]PAH(T)[], have shown the active site to be a six-coordinate distorted octahedral site. After the substrate and cofactor bind to the enzyme ([Fe(2+)]PAH(R)[L-Phe,5-deaza-6-MPH(4)]), the active site converts to a five-coordinate square pyramidal structure in which the identity of the missing ligand had not been previously determined. X-ray absorption spectroscopy (XAS) at the Fe K-edge further supports this coordination number change with the binding of both cosubstrates to the enzyme, and determines this to be due to the loss of a water ligand.
Animals↗
Structural characterization of metallopeptides designed as scaffolds for the stabilization of nickel(II)-Fe(4)S(4) bridged assemblies by X-ray absorption spectroscopy.
In earlier work, de novo designed peptides with a helix-loop-helix motif and 63 residues have been synthesized as potential scaffolds for stabilization of the [Ni(II)-X-Fe(4)S(4)] bridged assembly that is the spectroscopically deduced structure of the A-Cluster in clostridial carbon monoxide dehydrogenase. The 63mers contain a consensus tricysteinyl ferredoxin domain in the loop for binding an Fe(4)S(4) cluster and Cys and His residues proximate to the loop for binding Ni(II), with one Cys residue designed as the bridge X. The metallopeptides HC(4)H(2)-[Fe(4)S(4)]-Ni and HC(5)H-[Fe(4)S(4)]-M, containing three His and one Cys residue for Ni(II) coordination and two His and two Cys residues for binding M = Ni(II) and Co(II), have been examined by Fe-, Ni-, and Co-K edge spectroscopy and EXAFS. All peptides bind an [Fe(4)S(4)](2+) cubane-type cluster. Interpretation of the Ni and Co data is complicated by the presence of a minority population of six-coordinate species with low Z ligands, designated for simplicity as [M(OH(2))(6)](2+). Best fits of the data were obtained with ca. 20% [M(OH(2))(6)](2+) and ca. 80% M(II) with mixed N/S coordination. The collective XAS results for HC(4)H(2)-[Fe(4)S(4)]-Ni and HC(5)H-[Fe(4)S(4)]-M demonstrate the presence of an Fe(4)S(4) cluster and support the existence of the distorted square-planar coordination units [Ni(II)(S.Cys)(N.His)(3)] and [Ni(II)(S.Cys)(2)(N.His)(2)] in the HC(4)H(2) and HC(5)H metallopeptides, respectively. In the HC(5)H metallopeptide, tetrahedral [Co(II)(S.Cys)(2)(N.His)(2)] is present. We conclude that the designed scaffolded binding sites, including Ni-(mu(2)-S.Cys)-Fe bridges, have been achieved. This is the first XAS study of a de novo designed metallopeptide intended to stabilize a bridged biological assembly, and one of a few XAS analyses of metal derivatives of designed peptides. The scaffolding concept should be extendable to other bridged metal assemblies.
Aldehyde Oxidoreductases↗
Electronic structure description of the mu(4)-sulfide bridged tetranuclear Cu(Z) center in N(2)O reductase.
Spectroscopy coupled with density functional calculations has been used to define the spin state, oxidation states, spin distribution, and ground state wave function of the mu4-sulfide bridged tetranuclear CuZ cluster of nitrous oxide reductase. Initial insight into the electronic contribution to N2O reduction is developed, which involves a sigma superexchange pathway through the bridging sulfide.
Circular Dichroism↗
X-ray absorption spectroscopic investigation of Fe(II)-peplomycin and peplomycin derivatives: the effect of axial ligation on Fe-pyrimidine back-bonding.
X-ray absorption spectroscopy (XAS) is used to study ferrous complexes of a bleomycin (BLM) congener, peplomycin (PEP), and two of its derivatives, iso-peplomycin (ISO) and depyruvamide peplomycin (DP), in which potential axial ligands have been perturbed and removed, respectively. Application of extended X-ray absorption fine structure analysis shows an elongation of the short-distance component of the first coordination sphere in DP and ISO relative to PEP. The XAS pre-edge intensity concomitantly decreases with increased axial perturbation. The short-distance component of PEP is correlated to the Fe-pyrimidine bond and is related to the amount of pi-back-bonding. Thus, the XAS analysis of these complexes provides structural information relevant to their differences in O2 reactivity.
Anti-Bacterial Agents↗