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Britt Hedman

Publications and source records attributed to Britt Hedman.

At least 55 records · Page 3Linked to original sources
PubMed2003

Spectroscopic and electronic structure studies of 2,3-dihydroxybiphenyl 1,2-dioxygenase: O2 reactivity of the non-heme ferrous site in extradiol dioxygenases.

The extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHBD, EC 1.13.11.39), has been studied using magnetic circular dichroism (MCD), variable-temperature variable-field (VTVH) MCD, X-ray absorption (XAS) pre-edge, and extended X-ray absorption fine structure (EXAFS) spectroscopies, which are analogous to methods used in earlier studies on the extradiol dioxygenase catechol 2,3-dioxygenase [Mabrouk et al. J. Am. Chem Soc. 1991, 113, 4053-4061]. For DHBD, the spectroscopic data can be correlated to the results of crystallography and with the results from density functional calculations to obtain detailed geometric and electronic structure descriptions of the resting and substrate (DHB) bound forms of the enzyme. The geometry of the active site of the resting enzyme, square pyramidal with a strong Fe-glutamate bond in the equatorial plane, localizes the redox active orbital in an orientation appropriate for O(2) binding. However, the O(2) reaction is not favorable, as it would produce a ferric superoxide intermediate with a weak Fe-O bond. Substrate binding leads to a new square pyramidal structure with the strong Fe-glutamate bond in the axial direction as indicated by a decrease in the (5)E(g) and increase in the (5)T(2g) splitting. Electronic structure calculations provide insight into the relative lack of dioxygen reactivity for the resting enzyme and its activation upon substrate binding.

Binding Sites↗
PubMed2003

Spectroscopic investigation of stellacyanin mutants: axial ligand interactions at the blue copper site.

Detailed electronic and geometric structural descriptions of the blue copper sites in wild-type (WT) stellacyanin and its Q99M and Q99L axial mutants have been obtained using a combination of XAS, resonance Raman, MCD, EPR, and DFT calculations. The results show that the origin of the short Cu-S(Cys) bond in blue copper proteins is the weakened axial interaction, which leads to a shorter (based on EXAFS results) and more covalent (based on S K-edge XAS) Cu-S bond. XAS pre-edge energies show that the effective nuclear charge on the copper increases going from O(Gln) to S(Met) to no axial (Leu) ligand, indicating that the weakened axial ligand is not fully compensated for by the increased donation from the thiolate. This is further supported by EPR results. MCD data show that the decreased axial interaction leads to an increase in the equatorial ligand field, indicating that the site acquires a more trigonally distorted tetrahedral structure. These geometric and electronic structural changes, which result from weakening the bonding interaction of the axial ligand, allow the site to maintain efficient electron transfer (high H(DA) and low reorganization energy), while modulating the redox potential of the site to the biologically relevant range. These spectroscopic studies are complemented by DFT calculations to obtain insight into the factors that allow stellacyanin to maintain a trigonally distorted tetrahedral structure with a relatively strong axial Cu(II)-oxygen bond.

Binding Sites↗
PubMed2003

Spectroscopic studies of the interaction of ferrous bleomycin with DNA.

Bleomycin is an antibiotic used in cancer chemotherapy for its ability to achieve both single- and double-strand cleavage of DNA through abstraction of the deoxyribose C4'-H. Magnetic circular dichroism (MCD) and X-ray absorption (XAS) spectroscopies have been used to study the interaction of the biologically relevant FeIIBLM complex with DNA. Calf thymus DNA was used as the substrate as well as short oligonucleotides, including one with a preferred 5'-G-pyrimidine-3' cleavage site [d(GGAAGCTTCC)2] and one without [d(GGAAATTTCC)2]. DNA binding to FeIIBLM significantly perturbs the FeII active site, resulting in a change in intensity ratio of the d d transitions and a decrease in excited-state orbital splitting (5Eg). Although this effect is somewhat dependent on length and composition of the oligonucleotide, it is not correlated to the presence of a 5'-G-pyrimidine-3' cleavage site. No effect is observed on the charge-transfer transitions, indicating that the H-bonding recognition between the pyrimidine and guanine base does not perturb Fe-pyrimidine backbonding. Azide binding studies indicate that FeIIBLM bound to either oligomer has the same affinity for N3-. Parallel studies of BLM structural derivatives indicate that FeIIiso-PEPLM, in which the carbamoyl group is shifted on the mannose sugar, forms the same DNA-bound species as FeIIBLM. In contrast, FeIIDP-PEPLM, in which the -aminoalanine group is absent, forms a new species upon DNA binding. These data are consistent with a model in which the primary amine from the -aminoalanine is an FeII ligand and the mannose carbamoyl provides either a ligand to the FeII or significant second-sphere effects on the FeII site; intercalation of the bithiazole tail into the double helix likely brings the metal-bound complex close enough to the DNA to create steric interactions that remove the sugar groups from interaction with the FeII. The fact that the FeII active site is perturbed regardless of DNA sequence is consistent with the fact that cleavage is observed for both 5'-GC-3' and nonspecific oligomers and indicates that different reaction coordinates may be active, depending on orientation of the deoxyribose C4'-H.

Animals↗
PubMed2003

X-ray absorption spectroscopy of a structural analogue of the oxidized active sites in the sulfite oxidase enzyme family and related molybdenum(V) complexes.

X-ray absorption spectroscopy (XAS) (edge and extended X-ray absorption fine structure (EXAFS)) has been applied to the characterization of three molybdenum(V,VI) monodithiolene complexes with unidentate coligands, [MoO(SC(6)H(2)-2,4,6-Pr(i)()(3))(2)(bdt)](-) (1), [MoOCl(SC(6)H(2)-2,4,6-Pr(i)(3))(bdt)](-) (2), and [MoO(2)(SC(6)H(2)-2,4,6-Pr(i)(3))(bdt)](-) (3) (bdt = benzene-1,2-dithiolate). These complexes are related to the active site in the xanthine oxidase and sulfite oxidase families and, as in the enzyme sites, bind monodentate thiolate. By comparison to the data of crystalline oxidized chicken sulfite oxidase, it is shown that complex 3, whose thiolate simulates binding by the highly conserved cysteine, is an accurate structural analogue of the oxidized site of this enzyme. Normalized edge spectra, EXAFS data, Fourier transforms, and GNXAS-based fit results are presented. As in earlier studies, this provides characterization of new analogue complexes by XAS to facilitate identification of related sites in proteins.

Animals↗
PubMed2003

Description of the ground state wave functions of Ni dithiolenes using sulfur K-edge X-ray absorption spectroscopy.

The pterin-dithiolene cofactor is an essential component of the catalytic sites of all molybdoenzymes except nitrogenase. Understanding its bonding to transition metals allows for development of electronic structure/function correlations in catalysis. The electronic structure description for a series of bis(dithiolene) complexes ([NiL(2)](Z)(), L = 1,2-Me(2)C(2)S(2); Z = 2-, 1-, 0) using sulfur XAS provides the basis for extension to the biologically relevant metal-containing dithiolenes. The transition dipole integral has been developed for the dithiolene sulfur through correlation of XAS pre-edge energy positions of sulfide-, thiolate-, and enedithiolate-S. The ground state wave functions of all three NiL(2) complexes have more than 50% S character experimentally demonstrating the noninnocent behavior of the dithiolene ligand. The S K-edge experimental results are correlated with spin-unrestricted, broken-symmetry density functional calculations. These show only limited spin polarization in the neutral complex and delocalized, ligand based ground states for the mono- and dianionic complexes. These XAS and DFT results are correlated with other spectroscopic features and provide insight into reactivity.

Models, Molecular↗
PubMed2003

Spectroscopic and kinetic studies of PKU-inducing mutants of phenylalanine hydroxylase: Arg158Gln and Glu280Lys.

Phenylalanine hydroxylase (PAH) is a tetrahydrobiopterin-dependent, nonheme iron enzyme that catalyzes the hydroxylation of L-Phe to L-Tyr in the rate-limiting step of phenylalanine catabolism. This reaction is tightly coupled in the wild-type enzyme to oxidation of the tetrahydropterin cofactor. Dysfunction of PAH activity in humans leads to the disease phenylketonuria (PKU). We have investigated two PKU-inducing mutants, Arg158Gln and Glu280Lys, using kinetic methods, magnetic circular dichrosim (MCD) spectroscopy, and X-ray absorption spectroscopy (XAS). Analysis of the products produced by the mutant enzymes shows that although both oxidize pterin at more than twice the rate of wild-type enzyme, these reactions are only approximately 20% coupled to production of L-Tyr. Previous MCD and XAS studies had demonstrated that the resting Fe(II) site is six-coordinate in the wild-type enzyme and converts to a five-coordinate site when both L-Phe and reduced pterin are present in the active site. Although the Arg158Gln mutant forms the five-coordinate site when both cosubstrates are bound, the Fe(II) site of the Glu280Lys mutant remains six-coordinate. These results provide insight into the PAH reaction and disease mechanism at a molecular level, indicating that the first step of the mechanism is formation of a peroxy-pterin species, which subsequently reacts with the Fe(II) site if the pterin is properly oriented for formation of an Fe-OO-pterin bridge and an open coordination position is available on the Fe(II).

Absorptiometry, Photon↗
PubMed2003

Spectroscopic studies of the effect of ligand donor strength on the Fe-NO bond intradiol dioxygenases.

The geometric and electronic structure of NO bound to reduced protocatechuate 3,4-dioxygenase and its substrate (3,4-dihydroxybenzoate, PCA) complex have been examined by X-ray absorption (XAS), UV-vis absorption (Abs), magnetic circular dichroism (MCD), and variable temperature variable field (VTVH) MCD spectroscopies. The results are compared to those previously published on model complexes described as [FeNO]7 systems in which an S = 5/2 ferric center is antiferromagnetically coupled to an S = 1 NO-. XAS pre-edge analysis indicates that the Fe-NO units in FeIIIPCD[NO-] and FeIIIPCD[PCA,NO-] lack the greatly increased pre-edge intensity representative of most [FeNO]7 model sites. Furthermore, from extended X-ray absorption fine structure (EXAFS) analysis, the FeIIIPCD[NO-] and FeIIIPCD[PCA,NO-] active sites are shown to have an Fe-NO distance of at least 1.91 A, approximately 0.2 A greater than those found in the model complexes. The weakened Fe-NO bond is consistent with the overall lengthening of the bond lengths and the fact that VTVH MCD data show that NO(-)-->FeIII CT transitions are no longer polarized along the z-axis of the zero-field splitting tensor. The weaker Fe-NO bond derives from the strong donor interaction of the endogenous phenolate and substrate catecholate ligands, which is observed from the increased intensity in the CT region relative to that of [FeNO]7 model complexes, and from the shift in XAS edge position to lower energy. As NO is an analogue of O2, the effect of endogenous ligand donor strength on the Fe-NO bond has important implications with respect to O2 activation by non-heme iron enzymes.

Algorithms↗
PubMed2002

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↗
PubMed2002

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↗
PubMed2002

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↗
PubMed2002

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↗
PubMed2002

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↗
PubMed2002

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↗
PubMed2001

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↗
PubMed2001

A Short Copper-Copper Distance in a (µ-1,2-Peroxo)dicopper(II) Complex Having a 1,8-Naphthyridine Unit as an Additional Bridge This work was supported by grants from the National Science Foundation and the National Institutes of Health. We thank A. M. Barrios for help in acquiring resonance Raman spectra. X-ray absorption spectroscopic data were measured at SSRL. SSRL is funded by the Department of Energy, Office of Basic Energy Science. The SSRL Structural Molecular Biology Program is supported by the National Institute of Health, National Center for Research Resources, Biomedical Technology Program, and by the Department of Energy, Office of Biological and Environmental Research.

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Journal Article↗
PubMed1999

Relationship between the Dipole Strength of Ligand Pre-Edge Transitions and Metal-Ligand Covalency.

The electric dipole contributions to the observed pre-edge intensities in ligand K-edge X-ray absorption (XAS) spectra are analyzed in terms of covalent-bonding contributions between the metal and ligand for a prototype system with one hole in the d shell. One- and two-center contributions to the intensity are identified. By direct evaluation of the integrals involved in the intensity expression, the two-center terms are shown to be at least 1 order of magnitude smaller than the one-center terms and can be ignored to a reasonable approximation. The one-center terms reflect the amount of ligand character in the partially occupied metal-based MOs and are proportional to the intrinsic transition moment of a ligand-centered 1s --> np transition. The final intensity does not contain terms proportional to the square of the metal-ligand distance as might have been expected on the basis of the analogy between ligand K-edge and ligand-to-metal charge transfer (LMCT) transitions that both formally lead to transfer of electron density from the ligand to the metal. This is due to the fact that the transition density is completely localized on the ligand in the case of a ligand K-edge transition but is delocalized over the metal and the ligand in the case of a LMCT transition. The effective nuclear charge dependence of the one-center transition moment integral was studied by Hartree-Fock level calculations and was found to be small. Electronic relaxation effects were considered and found to be small from a Hartree-Fock calculation on a cupric chloride model.

Journal Article↗
PubMed1999

Spectroscopic Investigation of Reduced Protocatechuate 3,4-Dioxygenase: Charge-Induced Alterations in the Active Site Iron Coordination Environment.

Chemical reduction of the mononuclear ferric active site in the bacterial intradiol cleaving catecholic dioxygenase protocatechuate 3,4-dioxygenase (3,4-PCD, Brevibacterium fuscum) produces a high-spin ferrous center. We have applied circular dichroism (CD), magnetic circular dichroism (MCD), variable-temperature-variable-field (VTVH) MCD, X-ray absorption (XAS) pre-edge, and extended X-ray absorption fine structure (EXAFS) spectroscopies to investigate the geometric and electronic structure of the reduced iron center. Excited-state ligand field CD and MCD data indicate that the site is six-coordinate where the (5)E(g) excited-state splitting is 2033 cm(-)(1). VTVH MCD analysis of the ground state indicates that the site has negative zero-field splitting with a small rhombic splitting of the lowest doublet (delta = 1.6 +/- 0.3 cm(-)(1)). XAS pre-edge analysis also indicates a six-coordinate site while EXAFS analysis provides accurate bond lengths. Since previous spectroscopic analysis and the crystal structure of oxidized 3,4-PCD indicate a five-coordinate ferric active site, the results presented here show that the coordination number increases upon reduction. This is attributed to the coordination of a second solvent ligand. The coordination number increase relative to the oxidized site also appears to be associated with a large decrease in the ligand donor strength in the reduced enzyme due to protonation of the original hydroxide ligand.

Journal Article↗