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James E Penner-Hahn

Publications and source records attributed to James E Penner-Hahn.

17 recordsLinked to original sources
PubMed2006

Peptidic models for the binding of Pb(II), Bi(III) and Cd(II) to mononuclear thiolate binding sites.

Herein, we evaluate the binding of Pb(II) and Bi(III) to cysteine-substituted versions of the TRI peptides [AcG-(LKALEEK)4G-NH2] which have previously been shown to bind Hg(II) and Cd(II) in unusual geometries as compared with small-molecule thiol ligands in aqueous solutions. Studies of Pb(II) and Bi(III) with the peptides give rise to complexes consistent with the metal ions bound to three sulfur atoms with M-S distances of 2.63 and 2.54 A, respectively. Competition experiments between the metal ions Pb(II), Cd(II), Hg(II) and Bi(III) for the peptides show that Hg(II) has the highest affinity, owing to the initial formation of the extremely strong HgS2 bond. Cd(II) and Pb(II) have comparable binding affinities at pH > 8, while Bi(III) displays the weakest affinity, following the model, M(II) + (TRI LXC)3(3-) --> M(II)(TRI LXC)3(-). While the relevant equilibria for Hg(II) binding to the TRI peptides corresponds to a strong first step forming Hg(TRI LXC)2(HTRI LXC), followed by a single deprotonation to give Hg(TRI LXC)3(-), the binding of Cd(II) and Pb(II) is consistent with initial formation of M(II)(TRI LXC)(HTRI LXC)2 (+) at pH < 5 followed by a two-proton dissociation step (pK(a2)) yielding M(II)(TRI LXC)3(-). Pb(II)(TRI LXC)(HTRI LXC)2(+) converts to Pb(II)(TRI LXC)3(-) at slightly lower pH values than the corresponding Cd(II)-peptide complexes. In addition, Pb(II) displays a lower pK (a) of binding to the "d"-substituted peptide, (TRI L12C, pK(a2) = 12.0) compared with the "a"-substituted peptide, (TRI L16C, pK (a2) = 12.6), the reverse of the order seen for Hg(II) and Cd(II). Pb(II) also showed a stronger binding affinity for TRI L12C (K(bind) = 3.2 x 10(7) M(-1)) compared with that with TRI L16C (K(bind) = 1.2 x 10(7) M(-1)) at pH > 8.

Amino Acid Sequence↗
PubMed2005

Reexamination of lead(II) coordination preferences in sulfur-rich sites: implications for a critical mechanism of lead poisoning.

Recent studies suggest that the developmental toxicity associated with childhood lead poisoning may be attributable to interactions of Pb(II) with proteins containing thiol-rich structural zinc-binding sites. Here, we report detailed structural studies of Pb(II) in such sites, providing critical insights into the mechanism by which lead alters the activity of these proteins. X-ray absorption spectroscopy of Pb(II) bound to structural zinc-binding peptides reveals that Pb(II) binds in a three-coordinate Pb(II)-S(3) mode, while Zn(II) is known to bind in a four-coordinate mode in these proteins. This Pb(II)-S(3) coordination in peptides is consistent with a trigonal pyramidal Pb(II)-S(3) model compound previously reported by Bridgewater and Parkin, but it differs from many other reports in the small molecule literature which have suggested Pb(II)-S(4) as a preferred coordination mode for lead. Reexamination of the published structures of these "Pb(II)-S(4)" compounds reveals that, in almost all cases, the coordination number of Pb is actually 5, 6, or 8. The results reported herein combined with this new review of published structures suggest that lead prefers to avoid four-coordination in sulfur-rich sites, binding instead as trigonal pyramidal Pb(II)-S(3) or as Pb(II)-S(5-8). In the case of structural zinc-binding protein sites, the observation that lead binds in a three-coordinate mode, and in a geometry that is fundamentally different from the natural coordination of zinc in these sites, explains why lead disrupts the structure of these peptides and thus provides the first detailed molecular understanding of the developmental toxicity of lead.

Binding Sites↗
PubMed2005

A method for normalization of X-ray absorption spectra.

Accurate normalization of X-ray absorption data is essential for quantitative analysis of near-edge features. A method, implemented as the program MBACK, to normalize X-ray absorption data to tabulated mass absorption coefficients is described. Comparison of conventional normalization methods with MBACK demonstrates that the new normalization method is not sensitive to the shape of the background function, thus allowing accurate comparison of data collected in transmission mode with data collected using fluorescence ion chambers or solid-state fluorescence detectors. The new method is shown to have better reliability and consistency and smaller errors than conventional normalization methods. The sensitivity of the new normalization method is illustrated by analysis of data collected during an equilibrium titration.

Algorithms↗
PubMed2004

XANES evidence against a manganyl species in the S3 state of the oxygen-evolving complex.

Manganyl Mn=O species have been suggested as possible intermediates in photosynthetic water oxidation and as the reactive species in asymmetric olefin epoxidation. The first X-ray absorption spectrum for a MnV=O complex is reported. Comparison of the EXAFS data for Na[MnV=O(HMPAB) with those for lower-valent Mn complexes suggests that EXAFS measurements and edge-energy measurements are unlikely to have sufficient sensitivity to reliably reflect the presence of Mn=O species. In contrast, the preedge transition for Na[MnV=O(HMPAB) is 4-fold more intense than the most intense preedge transition observed for nonmanganyl complexes. This increase in intensity is shown to be sufficiently sensitive to allow detection of a manganyl species if it is formed. These data together with published data for the photosynthetic oxygen-evolving complex provide strong evidence against the presence of manganyl Mn=O species in the oxygen-evolving complex through the S3 state.

Manganese↗
PubMed2004

The first spectroscopic model for the S1 state multiline signal of the OEC.

The parallel-mode electron paramagnetic resonance (EPR) spectrum of the S(1) state of the oxygen-evolving complex (OEC) shows a multiline signal centered around g=12, indicating an integer spin system. The series of [Mn(2)(2-OHsalpn)(2)] complexes were structurally characterized in four oxidation levels (Mn(II)(2), Mn(II)Mn(III), Mn(III)(2), and Mn(III)Mn(IV)). By using bulk electrolysis, the [Mn(III)Mn(IV)(2-OHsalpn)(2)(OH)] is oxidized to a species that contains Mn(IV) oxidation state as detected by X-ray absorption near edge spectroscopy (XANES) and that can be formulated as Mn(IV)(4) tetramer. The parallel-mode EPR spectrum of this multinuclear Mn(IV)(4) complex shows 18 well-resolved hyperfine lines center around g=11 with an average hyperfine splitting of 36 G. This EPR spectrum is very similar to that found in the S(1) state of the OEC. This is the first synthetic manganese model complex that shows an S(1)-like multiline spectrum in parallel-mode EPR.

Electrochemistry↗
PubMed2004

An engineered pathway for the formation of protein disulfide bonds.

We have engineered a pathway for the formation of disulfide bonds. By imposing evolutionary pressure, we isolated mutations that changed thioredoxin, which is a monomeric disulfide reductase, into a [2Fe-2S] bridged dimer capable of catalyzing O2-dependent sulfhydryl oxidation in vitro. Expression of the mutant protein in Escherichia coli with oxidizing cytoplasm and secretion via the Tat pathway restored disulfide bond formation in strains that lacked the complete periplasmic oxidative machinery (DsbA and DsbB). The evolution of [2Fe-2S] thioredoxin illustrates how mutations within an existing scaffold can add a cofactor and markedly change protein function.

Amino Acid Motifs↗
PubMed2003

An atypical linear Cu(I)-S2 center constitutes the high-affinity metal-sensing site in the CueR metalloregulatory protein.

CueR is a copper-responsive genetic switch that regulates transcription of genes encoding the primary copper-exporting system in E. coli, CopA and CueO. Although a member of the MerR family of regulatory proteins, CueR has four cysteines in an array that is distinct from those in Hg(II)-sensing MerR or in Zn-sensing ZntR. A recent crystal structure showed one copper atom in CueR bound to cysteines Cys112 and Cys120 in a linear CuS2 structure, but left open the questions of whether the other half of the CueR dimer has the same structure, and of whether these structures depend on the two additional C-terminal cysteines. Metal binding, transcription runoff, and cysteine modification studies show that only Cys112 and Cys 120 are necessary and sufficient to make the transcriptionally active Cu-sensing site. X-ray absorption spectroscopy shows that this site binds Cu(I) in a strictly linear CuS2 site in solution, a structure that is rarely observed in copper proteins. This structure does not depend either on additional metal loading or upon the presence of additional C-terminal cysteine ligands and is well suited for an ultrasensitve receptor site that discriminates against the binding of other metal ions.

Bacterial Proteins↗
PubMed2003

Structural characterization of the zinc site in protein farnesyltransferase.

X-ray absorption spectroscopy has been used to determine the structure of the Zn site in protein farnesyltransferase. Extended X-ray absorption fine structure (EXAFS) data are consistent with a Zn site that is ligated to three low-Z (oxygen or nitrogen) ligands and one cysteine sulfur, as predicted from the crystal structures that are available for farnesyltransferase. However, in contrast with the crystallographic results the EXAFS data do not show evidence for significant distortions in the Zn-ligand distances. The average Zn-(N/O) and Zn-S distances are 2.04 and 2.31 A, respectively. Addition of a farnesyl diphosphate analogue causes no detectable change in the structure of the Zn site. However, addition of peptide substrate causes a change in ligation from ZnS(N/O)(3) to ZnS(2)(N/O)(2), consistent with ligation of the C-terminal cysteine to the Zn. There is no significant change in Zn-ligand distances when a substrate binds, demonstrating that the Zn remains four-coordinate. Addition of both peptide and farnesyl diphosphate to give the product complex causes the Zn to return to ZnS(N/O)(3) ligation, indicating that the product thioether is not tightly coordinated to the Zn. These spectroscopic experiments provide insight into the catalytic mechanism of FTase.

Alkyl and Aryl Transferases↗
PubMed2003

Models for the lower S states of photosystem II: a trinuclear mixed-valent MnII/MnIV/MnII complex.

The trinuclear complex Mn(3)(pko)(4)(CH(3)O)(2)(SCN)(2).CH(3)OH, 1, where Hpko is 2,2'-dipyridylketonoxime, is a rare example of a complex simultaneously containing Mn(II) and Mn(IV). X-ray crystallography and XANES spectroscopy clearly distinguish the Mn(II)(2)Mn(IV) valence isomer from the more commonly observed Mn(III)(2)Mn(II) formulation. Fits to variable-temperature magnetic susceptibility data indicate that the Mn(II) and Mn(IV) are ferromagnetically coupled (J = +6.13 cm(-1)) and that 1 has an S = (13)/(2) ground state.

Crystallography, X-Ray↗
PubMed2003

EXAFS studies of the zinc sites of UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC).

Extended X-ray absorption fine structure (EXAFS) spectroscopy has been used to determine the structure of the Zn(II) sites in UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC) from Aquifex aeolicus and Pseudomonas aeruginosa. The active site Zn(II) is four coordinate, with exclusively low-Z (nitrogen and oxygen) ligation in both enzymes. The amplitude of the outer-shell scattering from the histidine ligands is best fit using two histidine ligands, suggesting a ZnO(2)(His)(2) site, where O most likely represents a conserved aspartate and a solvent molecule. The same structure was found for Co(II)-substituted A. aeolicus LpxC, although in this case it is possible that the coordination sphere may expand to include a fifth low-Z ligand. EXAFS data were also measured for the Escherichia coli LpxC enzyme. When a single Co(II) is substituted for Zn(II) in the active site of E. coli LpxC, EXAFS data show the same ligand environment as is found for the P. aeruginosa and A. aeolicus enzymes. However, the EXAFS data for E. coli LpxC with two zinc ions bound per protein, with the second Zn(II) acting as an inhibitory metal, demonstrates that the inhibitory metal is bound to at least two high-Z (sulfur, presumably thiolate, or chlorine) ligands. Results of the outer-shell scattering analysis, combined with previous studies of the LpxC enzyme, indicate a novel zinc binding motif not found in any previously studied zinc metalloproteins.

Amidohydrolases↗
PubMed2003

The PcoC copper resistance protein coordinates Cu(I) via novel S-methionine interactions.

The E. coli copper resistance protein PcoC enhances survival of a bacterium under conditions of extreme copper stress. This small protein has no cysteines, but does have an unusual methionine-rich sequence motif, suggesting that methionine ligation may be important in Cu binding. It is shown that PcoC binds both Cu(I) and Cu(II), in addition to binding Hg(II) and Ag(I). Previously crystallographic studies of PcoC had shown that the apo protein adopts a beta-barrel fold typical of that seen for blue-copper electron-transfer proteins. However, in contrast with electron-transfer proteins, where the Cu(I) and Cu(II) structures are nearly identical, X-ray absorption spectra show that the structures of the Cu site in reduced and oxidized PcoC are dramatically different. Cu(II) PcoC has a tetragonal Cu structure in which the Cu is coordinated to O or N ligands, including at least two histidine ligands. Cu(I) PcoC has a trigonal site with two methionine ligands. This is the first well-characterized example of a methionine-rich protein Cu binding site, demonstrating a new type of biological Cu coordination chemistry.

Copper↗
PubMed2002

A light-dependent mechanism for massive accumulation of manganese in the photosynthetic bacterium Synechocystis sp. PCC 6803.

Manganese is an essential micronutrient for many organisms. Because of its unique role in the water oxidizing activity of photosystem II, manganese is required for photosynthetic growth in plants and cyanobacteria. Here we report on the mechanism of manganese uptake in the cyanobacterium Synechocystis sp. PCC 6803. Cells grown in 9 microM manganese-containing medium accumulate up to 1 x 10(8) manganese atoms/cell, bound to the outer membrane (pool A). This pool could be released by EDTA treatment. Accumulation of manganese in pool A was energized by photosynthetic electron flow. Moreover, collapsing the membrane potential resulted in the immediate release of this manganese pool. The manganese in this pool is mainly Mn(II) in a six-coordinate distorted environment. A distinctly different pool of manganese, pool B ( approximately 1.5 x 10(6) atoms/cell), could not be extracted by EDTA. Transport into pool B was light-independent and could be detected only under limiting manganese concentrations (1 nM). Evidently, manganese uptake in Synechocystis 6803 cells occurs in two steps. First, manganese accumulates in the outer membrane (pool A) in a membrane potential-dependent process. Next, manganese is transported through the inner membrane into pool B. We propose that pool A serves as a store that allows the cells to overcome transient limitations in manganese in the environment.

ATP-Binding Cassette Transporters↗
PubMed2002

Comparison of the binding of cadmium(II), mercury(II), and arsenic(III) to the de novo designed peptides TRI L12C and TRI L16C.

Designed alpha-helical peptides of the TRI family with a general sequence Ac-G(LKALEEK)(4)G-CONH(2) were used as model systems for the study of metal-protein interactions. Variants containing cysteine residues in positions 12 (TRI L12C) and 16 (TRI L16C) were used for the metal binding studies. Cd(II) binding was investigated, and the results were compared with previous and current work on Hg(II) and As(III) binding. The metal peptide assemblies were studied with the use of UV, CD, EXAFS, (113)Cd NMR, and (111m)Cd perturbed angular correlation spectroscopy. The metalated peptide aggregates exhibited pH-dependent behavior. At high pH values, Cd(II) was bound to the three sulfurs of the three-stranded alpha-helical coiled coils. A mixture of two species was observed, including Cd(II) in a trigonal planar geometry. The complexes have UV bands at 231 nm (20 600 M(-1) cm(-1)) for TRI L12C and 232 nm (22 600 M(-1) cm(-1)) for TRI L16C, an average Cd-S bond length of 2.49 A for both cases, and a (113)Cd NMR chemical shift at 619 ppm (Cd(II)(TRI L12C)(3)(-)) or 625 ppm (Cd(II)(TRI-L16C)(3)(-)). Nuclear quadrupole interactions show that two different Cd species are present for both peptides. One species with omega(0) = 0.45 rad/ns and low eta is attributed to a trigonal planar Cd-(Cys)(3) site. The other, with a smaller omega(0), is attributed to a four-coordinate Cd(Cys)(3)(H(2)O) species. At low pH, no metal binding was observed. Hg(II) binding to TRI L12C was also found to be pH dependent, and a 3:1 sulfur-to-mercury(II) species was observed at pH 9.4. These metal peptide complexes provide insight into heavy metal binding and metalloregulatory proteins such as MerR or CadC.

Amino Acid Sequence↗
PubMed2002

Elucidation of primary (alpha(3)N) and vestigial (alpha(5)) heavy metal-binding sites in Staphylococcus aureus pI258 CadC: evolutionary implications for metal ion selectivity of ArsR/SmtB metal sensor proteins.

Despite a common evolutionary origin, individual members of the ArsR/SmtB family of bacterial metal-responsive transcriptional repressors sense a wide range of heavy-metal ions. The molecular basis for this metal ion selectivity is unclear. Here, we establish that Staphylococcus aureus plasmid pI258 CadC, a Cd(II)/Pb(II)/Bi(III)/Zn(II) sensor, contains two distinct metal-binding sites: a thiolate-rich alpha(3)N site comprised exclusively of cysteine ligands that preferentially binds larger, softer metal ions such as Cd(II), Pb(II) and Bi(III); and a second C-terminal alpha(5) site, found at the dimer interface, that is devoid of cysteine ligands and preferentially binds smaller, harder metal ions [Co(II) and Zn(II)] concurrently with metal binding to the alpha(3)N site. Optical absorption and X-ray spectroscopies reveal that the alpha(3)N site can adopt distinct coordination geometries in order to accommodate different metal ions, i.e. Cd(II), Bi(III), Co(II) and Zn(II) form distorted tetrahedral S(4) complexes, while Pb(II) adopts a trigonal S(3) complex. Characterization of mutant CadCs reveals that the alpha(3)N site is composed of Cys58 and Cys60 from the alpha(3) helix of the helix-turn-helix DNA-binding domain and Cys7 and/or Cys11 from the N-terminal "arm" of CadC; Cys11 is excluded from the Pb(II) coordination sphere. Only the thiolate-rich alpha(3)N site is metalloregulatory for repressor binding to a fluorescein-labeled cad O/P oligonucleotide upon coordination to Cd(II), Pb(II), Bi(III), Zn(II), and weakly for Co(II). Substitution of Cys60 and Cys7 with non-ligating residues specifically abrogates metal-dependent negative regulation of cad O/P binding, despite the fact that C60G and C7G CadCs maintain high affinity for metals in altered coordination complexes. These findings reveal that formation of metal coordination bonds to Cys7 and Cys60 play primary roles in transducing the allosteric response in CadC. The evolutionary implications for metal ion selectivity of ArsR/SmtB metal sensor proteins are discussed.

Allosteric Regulation↗