Showing papers in "Journal of Biological Inorganic Chemistry in 2006"

Zinc-buffering capacity of a eukaryotic cell at physiological pZn.

Abstract: In spite of the paramount importance of zinc in biology, dynamic aspects of cellular zinc metabolism remain poorly defined at the molecular level. Investigations with human colon cancer (HT-29) cells establish a total cellular zinc concentration of 264 microM. Remarkably, about 10% of the potential high-affinity zinc-binding sites are not occupied by zinc, resulting in a surplus of 28 muM ligands (average Kd(c) = 83 pM) that ascertain cellular zinc-buffering capacity and maintain the "free" zinc concentration in proliferating cells at picomolar levels (784 pM, pZn = 9.1). This zinc-buffering capacity allows zinc to fluctuate only with relatively small amplitudes (DeltapZn = 0.3; below 1 nM) without significantly perturbing physiological pZn. Thus, the "free" zinc concentrations in resting and differentiated HT-29 cells are 614 pM and 1.25 nM, respectively. The calculation of these "free" zinc concentrations is based on measurements at different concentrations of the fluorogenic zinc-chelating agent and extrapolation to a zero concentration of the agent. It depends on the state of the cell, its buffering capacity, and the zinc dissociation constant of the chelating agent. Zinc induction of thionein (apometallothionein) ensures a surplus of unbound ligands, increases zinc-buffering capacity and the availability of zinc (DeltapZn = 0.8), but preserves the zinc-buffering capacity of the unoccupied high-affinity zinc-binding sites, perhaps for crucial physiological functions. Jointly, metallothionein and thionein function as the major zinc buffer under conditions of increased cellular zinc.

Metal ion ligands in hyperaccumulating plants

Abstract: Metal-hyperaccumulating plants have the ability to take up extraordinary quantities of certain metal ions without succumbing to toxic effects. Most hyperaccumulators select for particular metals but the mechanisms of selection are not understood at the molecular level. While there are many metal-binding biomolecules, this review focuses only on ligands that have been reported to play a role in sequestering, transporting or storing the accumulated metal. These include citrate, histidine and the phytosiderophores. The metal detoxification role of metallothioneins and phytochelatins in plants is also discussed.

The performance of hybrid DFT for mechanisms involving transition metal complexes in enzymes

Abstract: The accuracy of density functional theory with the B3LYP functional is reviewed for systems of relevance to transition-metal-containing enzymes. Calculated energies are commonly within 3-5 kcal/mol of the correct values; however, some exceptions have appeared in the literature and are discussed here. For example, the binding of NO and that of O(2) to metal centers have for some time been known to be underestimated. Most barriers for chemical reactions are overestimated except those involving hydrogen (or proton) transfer, which instead tend to be underestimated. A minor general improvement of the accuracy can probably be obtained by slightly reducing the amount of exact exchange in the B3LYP functional.

The active site of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans. II. Redox properties, light sensitivity and CO-ligand exchange as observed by infrared spectroscopy

Abstract: In [FeFe]-hydrogenases, the H cluster (hydrogen-activating cluster) contains a di-iron centre ([2Fe]H subcluster, a (L)(CO)(CN)Fe(mu-RS2)(mu-CO)Fe(CysS)(CO)(CN) group) covalently attached to a cubane iron-sulphur cluster ([4Fe-4S]H subcluster). The Cys-thiol functions as the link between one iron (called Fe1) of the [2Fe]H subcluster and one iron of the cubane subcluster. The other iron in the [2Fe]H subcluster is called Fe2. The light sensitivity of the Desulfovibrio desulfuricans enzyme in a variety of states has been studied with infrared (IR) spectroscopy. The aerobic inactive enzyme (H(inact) state) and the CO-inhibited active form (H(ox)-CO state) were stable in light. Illumination of the H(ox) state led to a kind of cannibalization; in some enzyme molecules the H cluster was destroyed and the released CO was captured by the H clusters in other molecules to form the light-stable H(ox)-CO state. Illumination of active enzyme under 13CO resulted in the complete exchange of the two intrinsic COs bound to Fe2. At cryogenic temperatures, light induced the photodissociation of the extrinsic CO and the bridging CO of the enzyme in the H(ox)-CO state. Electrochemical redox titrations showed that the enzyme in the H(inact) state converts to the transition state (H(trans)) in a reversible one-electron redox step (E (m, pH 7) = -75 mV). IR spectra demonstrate that the added redox equivalent not only affects the [4Fe-4S]H subcluster, but also the di-iron centre. Enzyme in the H(trans) state reacts with extrinsic CO, which binds to Fe2. The H(trans) state converts irreversibly into the H(ox) state in a redox-dependent reaction most likely involving two electrons (E (m, pH 7) = -261 mV). These electrons do not end up on any of the six Fe atoms of the H cluster; the possible destiny of the two redox equivalents is discussed. An additional reversible one-electron redox reaction leads to the H(red) state (E (m, pH 7) = -354 mV), where both Fe atoms of the [2Fe]H subcluster have the same formal oxidation state. The possible oxidation states of Fe1 and Fe2 in the various enzyme states are discussed. Low redox potentials (below -500 mV) lead to destruction of the [2Fe]H subcluster.

Transition metal spin state energetics and noninnocent systems: challenges for DFT in the bioinorganic arena

Abstract: Although density functional theory (DFT) provides a generally good description of transition metal systems, we have identified several cases, involving Fe(III) porphyrins and related systems, where common functionals fail to correctly describe the energetics of the different low-lying spin states. The question of metal- versus ligand-centered oxidation in high-valent transition metal complexes is also a challenging one for DFT calculations, as I have tried to illustrate with examples from among porphyrin, corrole, biliverdine, and NO complexes. In a number of cases, I have compared results obtained with different exchange–correlation functionals; in addition, I have added a discussion on the relative performance of pure versus hybrid functionals. Finally, I have offered some thoughts on the role that traditional wavefunction-based ab initio methods, now essentially absent from the bioinorganic arena, might play in the future.

Ferritins: iron/oxygen biominerals in protein nanocages.

Abstract: Ferritin protein nanocages that form iron oxy biominerals in the central nanometer cavity are nature's answer to managing iron and oxygen; gene deletions are lethal in mammals and render bacteria more vulnerable to host release of antipathogen oxidants. The multifunctional, multisubunit proteins couple iron with oxygen (maxi-ferritins) or hydrogen peroxide (mini-ferritins) at catalytic sites that are related to di-iron sites oxidases, ribonucleotide reductase, methane monooxygenase and fatty acid desaturases, and synthesize mineral precursors. Gated pores, distributed symmetrically around the ferritin cages, control removal of iron by reductants and chelators. Gene regulation of ferritin, long known to depend on iron and, in animals, on a noncoding messenger RNA (mRNA) structure linked in a combinatorial array to functionally related mRNA of iron transport, has recently been shown to be linked to an array of proteins for antioxidant responses such as thioredoxin and quinone reductases. Ferritin DNA responds more to oxygen signals, and ferritin mRNA responds more to iron signals. Ferritin genes (DNA and RNA) and protein function at the intersection of iron and oxygen chemistry in biology.

Perturbations of the T1 copper site in the CotA laccase from Bacillus subtilis: structural, biochemical, enzymatic and stability studies.

Abstract: Site-directed mutagenesis has been used to replace Met502 in CotA laccase by the residues leucine and phenylalanine. X-ray structural comparison of M502L and M502F mutants with the wild-type CotA shows that the geometry of the T1 copper site is maintained as well as the overall fold of the proteins. The replacement of the weak so-called axial ligand of the T1 site leads to an increase in the redox potential by approximately 100 mV relative to that of the wild-type enzyme (E 0=455 mV). However the M502L mutant exhibits a twofold to fourfold decrease in the k cat values for the all substrates tested and the catalytic activity in M502F is even more severely compromised; 10% activity and 0.15–0.05% for the non-phenolic substrates and for the phenolic substrates tested when compared with the wild-type enzyme. T1 copper depletion is a key event in the inactivation and thus it is a determinant of the thermodynamic stability of wild-type and mutant proteins. Whilst the unfolding of the tertiary structure in the wild-type enzyme is a two-state process displaying a midpoint at a guanidinium hydrochloride concentration of 4.6 M and a free-energy exchange in water of 10 kcal/mol, the unfolding for both mutant enzymes is clearly not a two-state process. At 1.9 M guanidinium hydrochloride, half of the molecules are in an intermediate conformation, only slightly less stable than the native state (approximately 1.4 kcal/mol). The T1 copper centre clearly plays a key role, from the structural, catalytic and stability viewpoints, in the regulation of CotA laccase activity.

Formate-reduced E. coli formate dehydrogenase H: the reinterpretation of the crystal structure suggests a new reaction mechanism

Abstract: Re-evaluation of the crystallographic data of the molybdenum-containing E. coli formate dehydrogenase H (Boyington et al. Science 275:1305-1308, 1997), reported in two redox states, reveals important structural differences for the formate-reduced form, with large implications for the reaction mechanism proposed in that work. We have re-refined the reduced structure with revised protocols and found substantial rearrangement in some parts of it. The original model is essentially correct but an important loop close to the molybdenum active site was mistraced, and, therefore, catalytic relevant residues were located in wrong positions. In particular selenocysteine-140, a ligand of molybdenum in the original work, and essential for catalysis, is no longer bound to the metal after reduction of the enzyme with formate. These results are incompatible with the originally proposed reaction mechanism. On the basis of our new interpretation, we have revised and proposed a new reaction mechanism, which reconciles the new X-ray model with previous biochemical and extended X-ray absorption fine structure data.

Structural and thermodynamical properties of CuII amyloid-β16/28 complexes associated with Alzheimer’s disease

Abstract: The aggregation of the peptide amyloid-β (Aβ) to form amyloid plaques is a key event in Alzheimer’s disease. It has been shown that CuII can bind to soluble Aβ and influence its aggregation properties. Three histidines and the N-terminal amine have been proposed to be involved in its coordination. Here, for the first time, we show isothermal titration calorimetry (ITC) measurements of the CuII binding to Aβ16 and Aβ28, models of the soluble Aβ. Moreover, different spectroscopic methods were applied. The studies revealed new insights into these CuII–Aβ complexes: (1) ITC showed two CuII binding sites, with an apparent Kd of 10−7 and 10−5 M, respectively; (2) the high-affinity site has a smaller enthalpic contribution but a larger entropic contribution than the low-affinity binding site; (3) azide did not bind to CuII in the higher-affinity binding site, suggesting the absence of a weak, labile ligand; (4) azide could bind to the CuII in the low-affinity binding site in Aβ28 but not in Aβ16; (5) 1H-NMR suggests that the carboxylate of aspartic acid in position 1 is involved in the ligation to CuII in the high-affinity binding site; (6) the pKa of 11.3 of tyrosine in position 10 was not influenced by the binding of 2 equivalents of CuII.

The active site of the [FeFe]-hydrogenase from Desulfovibrio desulfuricans . I. Light sensitivity and magnetic hyperfine interactions as observed by electron paramagnetic resonance

Abstract: The hydrogen-activating cluster (H cluster) in [FeFe]-hydrogenases consists of two moieties The [2Fe]H subcluster is a (L)(CO)(CN)Fe(μ-RS2)(μ-CO)Fe(CysS)(CO)(CN) centre The Cys-bound Fe is called Fe1, the other iron Fe2 The Cys-thiol forms a bridge to a [4Fe–4S] cluster, the [4Fe–4S]H subcluster We report that electron paramagnetic resonance (EPR) spectra of the 57Fe-enriched enzyme from Desulfovibrio desulfuricans in the Hox–CO state are consistent with a magnetic hyperfine interaction of the unpaired spin with all six Fe atoms of the H cluster In contrast to the inactive aerobic enzyme, the active enzyme is easily destroyed by light The [2Fe]H subcluster in some enzyme molecules loses CO by photolysis, whereupon other molecules firmly bind the released CO to form the Hox–CO state giving rise to the so-called axial 206 EPR signal Though not destroyed by light, the Hox–CO state is affected by it As demonstrated in the accompanying paper [49] two of the intrinsic COs, both bound to Fe2, can be exchanged by extrinsic 13CO during illumination at 2 °C We found that only one of the three 13COs, the one at the extrinsic position, gives an EPR-detectable isotropic superhyperfine interaction of 06 mT At 30 K both the inhibiting extrinsic CO bound to Fe2 and one more CO can be photolysed EPR spectra of the photolysed products are consistent with a 3d 7 system of Fe with the formal oxidation state +1 The damaged enzyme shows a light-sensitive g=5 signal which is ascribed to an S=3/2 form of the [2Fe]H subcluster The light sensitivity of the enzyme explains the occurrence of the g=5 signal and the axial 206 signal in published EPR spectra of nearly all preparations studied thus far

A single-crystal ENDOR and density functional theory study of the oxidized states of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F

Abstract: The catalytic center of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F in the oxidized states was investigated by electron paramagnetic resonance and electron–nuclear double resonance spectroscopy applied to single crystals of the enzyme. The experimental results were compared with density functional theory (DFT) calculations. For the Ni-B state, three hyperfine tensors could be determined. Two tensors have large isotropic hyperfine coupling constants and are assigned to the β-CH2 protons of the Cys-549 that provides one of the bridging sulfur ligands between Ni and Fe in the active center. From a comparison of the orientation of the third hyperfine tensor with the tensor obtained from DFT calculations an OH− bridging ligand has been identified in the Ni-B state. For the Ni-A state broader signals were observed. The signals of the third proton, as observed for the “ready” state Ni-B, were not observed at the same spectral position for Ni-A, confirming a structural difference involving the bridging ligand in the “unready” state of the enzyme.

Synthesis, characterization, antitumoral and osteogenic activities of quercetin vanadyl(IV) complexes

Abstract: The development of new vanadium derivatives with organic ligands, which improve the beneficial actions (insulin-mimetic, antitumoral) and decrease the toxic effects, is of great interest. A good candidate for the generation of a new vanadium compound is the flavonoid quercetin because of its own anticarcinogenic effect. The complex [VO(Quer)2EtOH] n (QuerVO) has been synthesized and characterized by means of different spectroscopic techniques (UV–vis, Fourier transform IR, electron paramagnetic resonance) and its magnetic and stability properties. The inhibitory effect on bovine alkaline phosphatase (ALP) activity has been tested for the free ligand, the complex as well as for the vanadyl(IV) (comparative purposes). The biological activity of the complex on the proliferation of two osteoblast-like cells in culture, a normal one (MC3T3E1) and a tumoral one (UMR106), has been compared with that of the vanadyl(IV) cation and quercetin. The differentiation osteoblast markers ALP specific activity and collagen synthesis have been also tested. In addition, the effect of QuerVO on the activation of the extracellular regulated kinase (ERK) pathway is reported. The bone antitumoral effect of quercetin alone was established with the cell proliferation assays (it inhibits the proliferation of the tumoral cells and does not exert any effect on the normal osteoblasts). Moreover, the complex exerts osteogenic effects since it stimulates the type I collagen production and is a weak inhibitory agent upon ALP activity. Finally, QuerVO stimulated the ERK phosphorylation in a dose–response manner and this activation seems to be involved as one of the possible mechanisms for the biological effects of the complex.

Reduction of dioxygen by enzymes containing copper

Abstract: The reduction of dioxygen is a key step in many important biological processes including respiration and ligand oxidation. Enzymes containing either iron or copper or, indeed, both elements are often involved in this process, yet the catalytic mechanisms employed are not fully understood at the current time despite intensive biochemical, spectroscopic and structural studies. The aim of this article is to highlight the current structural knowledge regarding the process of dioxygen reduction using examples of copper-containing enzymes.

Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego

Abstract: The membrane-bound human cytochrome P450s have essential roles in the metabolism of endogenous compounds and drugs. Presented here are the results on the construction and characterization of three fusion proteins containing the N-terminally modified human cytochrome P450s CYP2C9, CY2C19 and CYP3A4 fused to the soluble NADPH-dependent oxidoreductase domain of CYP102A1 from Bacillus megaterium. The constructs, CYP2C9/BMR, CYP2C19/BMR and CYP3A4/BMR are well expressed in Escherichia coli as holo proteins. The chimeras can be purified in the absence of detergent and the purified enzymes are both active and correctly folded in the absence of detergent, as demonstrated by circular dichroism and functional studies. Additionally, in comparison with the parent P450 enzyme, these chimeras have greatly improved solubility properties. The chimeras are catalytically self-sufficient and present turnover rates similar to those reported for the native enzymes in reconstituted systems, unlike previously reported mammalian cytochrome P450 fusion proteins. Furthermore the specific activities of these chimeras are not dependent on the enzyme concentration present in the reaction buffer and they do not require the addition of accessory proteins, detergents or phospholipids to be fully active. The solubility, catalytic self-sufficiency and wild-type like activities of these chimeras would greatly simplify the studies of cytochrome P450 mediated drug metabolism in solution.

Structure, redox, pKa, spin. A golden tetrad for understanding metalloenzyme energetics and reaction pathways.

Abstract: After a review of the current status of density functional theory (DFT) for spin-polarized and spin-coupled systems, we focus on the resting states and intermediates of redox-active metalloenzymes and electron transfer proteins, showing how comparisons of DFT-calculated spectroscopic parameters with experiment and evaluation of related energies and geometries provide important information. The topics we examine include (1) models for the active-site structure of methane monooxygenase intermediate Q and ribonucleotide reductase intermediate X; (2) the coupling of electron transfer to proton transfer in manganese superoxide dismutase, with implications for reaction kinetics; (3) redox, pK a, and electronic structure issues in the Rieske iron–sulfur protein, including their connection to coupled electron/proton transfer, and an analysis of how partial electron delocalization strongly alters the electron paramagnetic resonance spectrum; (4) the connection between protein-induced structural distortion and the electronic structure of oxidized high-potential 4Fe4S proteins with implications for cluster reactivity; (5) an analysis of cluster assembly and central-atom insertion into the FeMo cofactor center of nitrogenase based on DFT structural and redox potential calculations.

Heterocyclic zinc-binding groups for use in next-generation matrix metalloproteinase inhibitors: potency, toxicity, and reactivity

Abstract: In an effort to improve the zinc-chelating portion of matrix metalloproteinase (MMP) inhibitors, we have developed a family of heterocyclic zinc-binding groups (ZBGs) as alternatives to the widely used hydroxamic acid moiety Elaborating on findings from an earlier report, we performed in vitro inhibition assays with recombinant MMP-1, MMP-2, and in a cell culture assay using neonatal rat cardiac fibroblast cells In both recombinant and cell culture assays, the new ZBGs were found to be effective inhibitors, typically 10-100-fold more potent than acetohydroxamic acid The toxicity of these chelators was examined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt cytotoxicity assays, which demonstrate that most of these compounds are nontoxic at concentrations of almost 100 microM To address the possible interaction of sulfur-containing ZBGs with biological reductants, the reactivity of these chelators with 5,5'-dithiobis(2-nitrobenzoic acid) was examined Finally, thione ZBGs were shown to be effective inhibitors of cell invasion through an extracellular matrix membrane The data presented herein suggest these heterocyclic ZBGs are potent, nontoxic, and biocompatible compounds that show promise for incorporation into a new family of MMP inhibitors

Density functional study of the catalytic cycle of nickel–iron [NiFe] hydrogenases and the involvement of high-spin nickel(II)

Abstract: In light of recent experiments suggesting high-spin (HS) Ni(II) species in the catalytic cycle of [NiFe] hydrogenase, a series of models of the Ni(II) forms Ni-SI(I,II), SI-CO and Ni-R(I,II,III) were examined in their high-spin states via density functional calculations. Because of its importance in the catalytic cycle, the Ni–C form was also included in this study. Unlike the Ni(II) forms in previous studies, in which a low-spin (LS) state was assumed and a square–planar structure found, the optimized geometries of these HS Ni(II) forms resemble those observed in the crystal structures: a distorted tetrahedral to distorted pyramidal coordination for the NiS4. This resemblance is particularly significant because the LS state is 20–30 kcal/mol less stable than the HS state for the geometry of the crystal structure. If these Ni(II) forms in the enzyme are not high spin, a large change in geometry at the active site is required during the catalytic cycle. Furthermore, only the HS state for the CO-inhibited form SI-CO has CO stretching frequencies that match the experimental results. As in the previous work, these new results show that the heterolytic cleavage reaction of dihydrogen (where H2 is cleaved with the metal acting as a hydride acceptor and a cysteine as the proton acceptor) has a lower energy barrier and is more exothermic when the active site is oxidized to Ni(III). The enzyme models described here are supported by a calibrated correlation of the calculated and measured CO stretching frequencies of the forms of the enzyme. The correlation coefficient for the final set of models of the forms of [NiFe] hydrogenase is 0.8.

DNA binding properties of novel cytotoxic gold(III) complexes of terpyridine ligands: the impact of steric and electrostatic effects.

Abstract: Four gold(III) complexes of terpyridine derivatives 1-4 have been synthesized and characterized by spectroscopic methods. In vitro data demonstrated that all of them showed higher cytotoxicity than cisplatin against the human non-small-cell lung cancer cell line (A-549), the human stomach carcinoma cell line (SGC-7901), the human cervix carcinoma cell line (HELA), the human colon carcinoma cell line (HCT-116), the human liver carcinoma cell line (BEL-7402), the murine leukemia cell line (P-388) and the human acute promyelocytic leukemia cell line (HL-60). Complex 3 exhibits the highest activity, with growth inhibition rates of over 80% at 10(-8) mol L(-1) against the A-549, HCT-116 and HELA tumor cell lines. Interestingly, ligands L1-L4 are also very cytotoxic against the cell lines tested. Complexes 1-4 are stable in aqueous solution for 2 days in the presence of the biological reducing agent glutathione. The inductively coupled plasma mass spectrometry data showed that DNA isolated from cells treated with complexes 1 and 3 contained gold with gold-to-nucleotide ratios of approximately 1:6,400 and 1:4,900, respectively. Fluorescence titration, UV and circular dichroism analyses proved that the steric and electrostatic effects of the ligand remarkably influence the interactions of their gold(III) complexes with DNA. The DNA binding ability of the complexes has been correlated with their cytotoxicity, which could potentially provide a new rationale for the future design of terpyridine-based metal complexes with antitumor potential.

Structure and membrane affinity of new amphiphilic siderophores produced by Ochrobactrum sp. SP18

Abstract: The coastal α-proteobacterium Ochrobactrum sp. SP18 produces a suite of three citrate-derived, cell-associated amphiphilic siderophores, ochrobactins A–C. The ochrobactins are composed of a citric acid backbone amide-linked to two lysine residues. Each e-amine of lysine is hydroxylated and acylated forming two hydroxamic acid moieties. One of the acylated appendages of each ochrobactin is (E)-2-decenoic acid. The other acylated appendages for ochrobactins A–C are (E)-2-octenoic acid, octanoic acid and (E)-2-decenoic acid, respectively. The ferric ochrobactin complexes are photoreactive in UV light, producing an oxidized ligand with loss of 46 mass units that can still coordinate Fe(III). The relative partitioning of the apo-ochrobactins, Fe(III) ochrobactins and Fe(III) photoproducts into 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles is presented. The ochrobactins are the first example of aerobactin-based siderophores with two fatty acid appendages produced in a suite with varying acyl appendage lengths.

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

Abstract: 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)33− → 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 8.

Crystal structure of yeast Sco1

Abstract: The Sco family of proteins are involved in the assembly of the dinuclear CuA site in cytochrome c oxidase (COX), the terminal enzyme in aerobic respiration. These proteins, which are found in both eukaryotes and prokaryotes, are characterized by a conserved CXXXC sequence motif that binds copper ions and that has also been proposed to perform a thiol:disulfide oxidoreductase function. The crystal structures of Saccharomyces cerevisiae apo Sco1 (apo-ySco1) and Sco1 in the presence of copper ions (Cu–ySco1) were determined to 1.8- and 2.3-A resolutions, respectively. Yeast Sco1 exhibits a thioredoxin-like fold, similar to that observed for human Sco1 and a homolog from Bacillus subtilis. The Cu–ySco1 structure, obtained by soaking apo-ySco1 crystals in copper ions, reveals an unexpected copper-binding site involving Cys181 and Cys216, cysteine residues present in ySco1 but not in other homologs. The conserved CXXXC cysteines, Cys148 and Cys152, can undergo redox chemistry in the crystal. An essential histidine residue, His239, is located on a highly flexible loop, denoted the Sco loop, and can adopt positions proximal to both pairs of cysteines. Interactions between ySco1 and its partner proteins yeast Cox17 and yeast COX2 are likely to occur via complementary electrostatic surfaces. This high-resolution model of a eukaryotic Sco protein provides new insight into Sco copper binding and function.

Using synthetic chemistry to understand copper protein active sites: a personal perspective.

Abstract: The results of studies performed in the author’s laboratory are surveyed, with particular emphasis on demonstrating the value of a multidisciplinary synthetic modeling approach for discovering new and unusual chemistry helpful for understanding the properties of the active sites of copper proteins or assessing the feasibility of mechanistic pathways they might follow during catalysis. The discussion focuses on the progress made to date toward comprehending the nitrite reductase catalytic site and mechanism, the electronic structures of copper thiolate electron transfer centers, the sulfido-bridged “CuZ” site in nitrous oxide reductase, and the processes of dioxygen binding and activation by mono- and dicopper centers in oxidases and oxygenases.

Conformational transitions and redox potential shifts of cytochrome P450 induced by immobilization.

Abstract: Cytochrome P450 (P450) from Pseudomonas putida was immobilized on Ag electrodes coated with self-assembled monolayers (SAMs) via electrostatic and hydrophobic interactions as well as by covalent cross-linking. The redox and conformational equilibria of the immobilized protein were studied by potential-dependent surface-enhanced resonance Raman spectroscopy. All immobilization conditions lead to the formation of the cytochrome P420 (P420) form of the enzyme. The redox potential of the electrostatically adsorbed P420 is significantly more positive than in solution and shows a steady downshift upon shortening of the length of the carboxyl-terminated SAMs, i.e., upon increasing the strength of the local electric field. Thus, two opposing effects modulate the redox potential of the adsorbed enzyme. First, the increased hydrophobicity of the heme environment brought about by immobilization on the SAM tends to upshift the redox potential by stabilizing the formally neutral ferrous form. Second, increasing electric fields tend to stabilize the positively charged ferric form, producing the opposite effect. The results provide insight into the parameters that control the structure and redox properties of heme proteins and contribute to the understanding of the apparently anomalous behavior of P450 enzymes in bioelectronic devices.

Decomposition of reactive oxygen species by copper(II) bis(1-pyrazolyl)methane complexes

Abstract: Two bis(1-pyrazolyl)alkane ligands, bis(3,5-dimethyl-1-pyrazolyl)methane and bis(4-iodo-3,5-dimethyl-1-pyrazolyl)methane, and their copper(II) complexes, bis(3,5-dimethyl-1-pyrazolyl)methanedinitratocopper(II) [CuL1(NO3)2] and bis(4-iodo-3,5-dimethyl-1-pyrazolyl)methanedinitratocopper(II) [CuL2(NO3)2]·2H2O, were prepared. Physiochemical properties of the copper(II) complexes were studied by spectroscopic (UV–vis, IR, EPR) techniques and cyclic voltammetry. Spectroscopic analysis revealed a 1:1 stoichiometry of ligand:copper(II) ion and a bindentate coordination mode for the nitrate ions in both of the complexes. According to experimental and theoretical ab initio data, the copper(II) ion is located in an octahedral hexacoordinated environment. Both complexes were able to catalyze the dismutation of superoxide anion (\( {\text{O}}^{{\bullet - }}_{{\text{2}}} \)) (pH 7.5) and decomposition of H2O2 (pH 7.5) and peroxynitrite (pH 10.9). In addition, both complexes exhibited superoxide dismutase (SOD) like activity toward extracellular and intracellular reactive oxygen species produced by activated human neutrophils in whole blood. Thus, these complexes represent useful SOD mimetics with a broad range of antioxidant activity toward a variety of reactive oxidants.

Structural studies of constitutive nitric oxide synthases with diatomic ligands bound.

Abstract: Crystal structures are reported for the endothelial nitric oxide synthase (eNOS)–arginine–CO ternary complex as well as the neuronal nitric oxide synthase (nNOS) heme domain complexed with l-arginine and diatomic ligands, CO or NO, in the presence of the native cofactor, tetrahydrobiopterin, or its oxidized analogs, dihydrobiopterin and 4-aminobiopterin. The nature of the biopterin has no influence on the diatomic ligand binding. The binding geometries of diatomic ligands to nitric oxide synthase (NOS) follow the {MXY} n formalism developed from the inorganic diatomic–metal complexes. The structures reveal some subtle structural differences between eNOS and nNOS when CO is bound to the heme which correlate well with the differences in CO stretching frequencies observed by resonance Raman techniques. The detailed hydrogen-bonding geometries depicted in the active site of nNOS structures indicate that it is the ordered active-site water molecule rather than the substrate itself that would most likely serve as a direct proton donor to the diatomic ligands (CO, NO, as well as O2) bound to the heme. This has important implications for the oxygen activation mechanism critical to NOS catalysis.

EF-hand protein dynamics and evolution of calcium signal transduction: an NMR view

Abstract: Calcium signaling, one of the most widespread signaling mechanisms in cells, is generally carried out by EF-hand proteins, characterized by a helix–loop–helix motif paired in functional domains. EF-hand proteins may be viewed as molecular switches activated by calcium concentration transients. The EF-hand structural database has grown to a point where meaningful inferences on the functional conformational rearrangements upon calcium binding can be made by comparing a fair number of pairs of end points, i.e., the structures of the apo and calcium-bound forms. More compact descriptors of the movement associated with calcium binding, in terms of principal component analysis of the six interhelical angles, have also become available. Dynamic information obtained by NMR, also with the aid of calcium substitution with paramagnetic lanthanides, is shedding light on the intrinsic amplitude of the conformational degrees of freedom sampled by the various members of the EF-hand superfamily, as well as on the time scales of the motions. Particularly, NMR of lanthanide derivatives helps in capturing long time scale motions. Both static and dynamic pictures reveal a large variety of behaviors. It is increasingly recognized that the EF-hand machinery has differentiated its behavior during evolution in several ways, e.g., by modifying one of the loops, by undergoing a further duplication after the initial motif duplication that originated the functional domain, or by acquiring the ability to dimerize.

Bacterial siderophores: the solution stoichiometry and coordination of the Fe(III) complexes of pyochelin and related compounds

Abstract: Pyochelin, its analog 3''-nor-NH-pyochelin, and the related methyl hydroxamate, 2-(2'-hydroxyphenyl)-4,5-dihydrothiazol-4-carboxylic acid methoxymethyl amide, have been prepared together with their Fe(III) complexes. The solution stoichiometry and the coordination of the three Fe(III) complexes in methanol or buffered (pH approximately 2) 50:50 (v/v) methanol-water mixtures were determined using various spectroscopic methods: UV-vis absorption, X-ray absorption, extended X-ray absorption fine structure and electron paramagnetic resonance. All three systems showed both a 1:1 and 2:1 ligand-Fe(III) stoichiometry, but presented different coordination properties. Conditional formation constants (pH approximately 2) were determined for both the 1:1 and 2:1 complexes in all three systems. Computation of the coordination-conformational energies by semiempirical methods indicated that the coordination in the case of the 2:1 complexes of pyochelin-Fe(III) and 3''-nor-NH-pyochelin-Fe(III) was asymmetrical, with one molecule of pyochelin (or 3''-nor-NH-pyochelin) tetradentately coordinated (O1, N1, N2 and O3) to the Fe(III), and the second molecule bound bidentately (O1, N1 or N2, O3), to complete the octahedral geometry. In contrast, two molecules of the methyl hydroxamate each provided a set of tridentate ligand atoms in the formation of the 2:1 ligand-Fe(III) complex. These results are consistent with the role of pyochelin in the uptake of iron by the FptA receptor in the outer membrane of Pseudomonas aeruginosa and in several gram-negative bacteria.

Catalysis of regioselective reduction of NAD + by ruthenium(II) arene complexes under biologically relevant conditions

Abstract: Ruthenium(II) arene anticancer complexes [(η 6-arene)Ru(en)Cl]PF6 (arene is hexamethylbenzene, p-cymene, indan; en is ethylenediamine) can catalyse regioselective reduction of NAD+ by formate in water to form 1,4-NADH, at pD 7.2, 37 °C, and in the presence of air. The catalytic activity is markedly dependent on the arene, with the hexamethylbenzene (hmb) complex showing the highest activity. For [(η 6-hmb)Ru(en)Cl]PF6, the rate of reaction is independent of NAD+ concentration and shows saturation kinetics with respect to formate concentration. A K m value of 58 mM and a turnover frequency at saturation of 1.46 h−1 were observed. Removal of chloride and performing the reaction under argon led to higher reaction rates. Lung cancer cells (A549) were found to be remarkably tolerant to formate even at millimolar concentrations. The possibility of using ruthenium arene complexes coadministered with formate as catalytic drugs is discussed.

A spectroscopic and voltammetric study of the pH-dependent Cu(II) coordination to the peptide GGGTH: relevance to the fifth Cu(II) site in the prion protein.

Abstract: The GGGTH sequence has been proposed to be the minimal sequence involved in the binding of a fifth Cu(II) ion in addition to the octarepeat region of the prion protein (PrP) which binds four Cu(II) ions. Coordination of Cu(II) by the N- and C-protected Ac-GGGTH-NH2 pentapeptide (P5) was investigated by using potentiometric titration, electrospray ionization mass spectrometry, UV–vis spectroscopy, electron paramagnetic resonance (EPR) spectroscopy and cyclic voltammetry experiments. Four different Cu(II) complexes were identified and characterized as a function of pH. The Cu(II) binding mode switches from NO3 to N4 for pH values ranging from 6.0 to 10.0. Quasi-reversible reduction of the [CuII(P5)H−2] complex formed at pH 6.7 occurs at E 1/2=0.04 V versus Ag/AgCl, whereas reversible oxidation of the [CuII(P5)H−3]− complex formed at pH 10.0 occurs at E 1/2=0.66 V versus Ag/AgCl. Comparison of our EPR data with those of the rSHaPrP(90-231) (Burns et al. in Biochemistry 42:6794–6803, 2003) strongly suggests an N3O binding mode at physiological pH for the fifth Cu(II) site in the protein.