JBIC Journal of Biological Inorganic Chemistry (v.13, #1)
Letter from the Editor by Lawrence Que Jr (1-1).
Metals in the “omics” world: copper homeostasis and cytochrome c oxidase assembly in a new light by Ivano Bertini; Gabriele Cavallaro (3-14).
System-level understanding of living organisms has been a long-standing goal of biological sciences. However, it was only recently that this possibility became concrete, by virtue of the development of technology platforms for the production of “omics” data from multiple experimental sources. Data sets such as those from genomics and proteomics are endowing researchers with an unprecedented view of the molecular constituents of cells and of their interactions, forming the basis to pursue the comprehension of how the concerted action of such components can determine biological functions. Within this challenge, bioinorganic chemistry is invested with a renewed significance, being called to place its distinctive subject matter, namely, the study of the interactions between inorganic and biological molecules, in a system-wide perspective. The first step to take in this direction is the construction of “omics” data sets for metalloproteins (metalloproteomics) that can be fruitfully integrated with other protein-centered “omics” data. While looking forward to the progress of high-throughput experimental techniques to accomplish this task, theoretical methods are yielding valuable predictions as to the number of metalloproteins encoded in various genomes. The integrated use of these and others “omics” data can be extremely useful to model complex cellular processes involving metals. Here, we review the current knowledge on copper homeostasis and the assembly of cytochrome c oxidase to exemplify the kind of important processes which need to be studied at the system level. The long-term goal of this approach is the overall description of how metals are framed as essential factors within living cells, which in fact is the ultimate purpose of bioinorganic chemistry.
Keywords: Metalloproteomics; Systems biology; Metals; Copper; Cytochrome c oxidase
Spin concentration measurements of high-spin (g′ = 4.3) rhombic iron(III) ions in biological samples: theory and application by Fadi Bou-Abdallah; N. Dennis Chasteen (15-24).
Electron paramagnetic resonance (EPR) signals at g′ = 4.3 are commonly encountered in biological samples owing to mononuclear high-spin (S = 5/2) Fe3+ ions in sites of low symmetry. The present study was undertaken to develop the experimental method and a suitable g′ = 4.3 intensity standard and for accurately quantifying the amount of Fe3+ responsible for such signals. By following the work of Aasa and Vänngård (J. Magn. Reson. 19:308–315, 1975), we present equations relating the EPR intensity of S = 5/2 ions to the intensities of S = 1/2 standards more commonly employed in EPR spectrometry. Of the chelates tested, Fe3+–EDTA (1:3 ratio) in 1:3 glycerol/water (v/v), pH 2, was found to be an excellent standard for frozen-solution S = 5/2 samples at 77 K. The spin concentrations of Cu2+–EDTA and aqua VO2+, both S = 1/2 ions, and of Fe3+–transferrin, an S = 5/2 ion, were measured against this standard and found to agree within 2.2% of their known metal ion concentrations. Relative standard deviations of ±3.6, ±5.3 and ±2.9% in spin concentration were obtained for the three samples, respectively. The spin concentration determined for Fe3+–desferrioxamine of known Fe3+ concentration was anomalously low suggesting the presence of EPR-silent multimeric iron species in solution.
Keywords: Spin concentrations; Iron electron paramagnetic resonance; Siderophores; Mononuclear iron; Non-heme iron proteins
Characterization of N-terminal amino group–heme ligation emerging upon guanidine hydrochloric acid induced unfolding of Hydrogenobacter thermophilus ferricytochrome c 552 by Hulin Tai; Shin Kawano; Yasuhiko Yamamoto (25-34).
Nonnative heme coordination structures emerging upon guanidine hydrochloric acid (GdnHCl) induced unfolding of Hydrogenobacter thermophilus ferricytochrome c 552 were characterized by means of paramagnetic NMR. The heme coordination structure possessing the N-terminal amino group of the peptide chain in place of axial Met (His–Nterm form) was determined in the presence of GdnHCl concentrations in excess of 1.5 M at neutral pH. The stability of the His–Nterm form at pH 7.0 was found to be comparable with that of the bis-His form which has been recognized as a major nonnative heme coordination structure in cytochrome c folding/unfolding. Consequently, in addition to the bis-His form, the His–Nterm form is a substantial intermediate which affects the pathway and kinetics of the folding/unfolding of cytochromes c, of which the N-terminal amino groups are not acetylated.
Keywords: Cytochrome; Electronic structure; Ligand binding; Protein folding; Site-directed mutagenesis
Towards extracellular Ca2+ sensing by MRI: synthesis and calcium-dependent 1H and 17O relaxation studies of two novel bismacrocyclic Gd3+ complexes by Kirti Dhingra; Petra Fousková; Goran Angelovski; Martin E. Maier; Nikos K. Logothetis; Éva Tóth (35-46).
Two new bismacrocyclic Gd3+ chelates containing a specific Ca2+ binding site were synthesized as potential MRI contrast agents for the detection of Ca2+ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca2+-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L1) or by a propylene (L2) unit [H4BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H3DO3A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd3+ complexes towards Ca2+ and Mg2+ was studied by 1H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca2+ binding to Gd2L1 and Gd2L2, respectively, with a distinct selectivity of Gd2L1 towards Ca2+ compared with Mg2+. For Ca2+ binding, association constants of log K = 1.9 (Gd2L1) and log K = 2.7 (Gd2L2) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu3+ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca2+ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca2+ addition. A 1H nuclear magnetic relaxation dispersion and 17O NMR study on Gd2L1 in the absence and in the presence of Ca2+ was performed to assess the microscopic parameters influencing relaxivity. On Ca2+ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction.
Keywords: Relaxivity; MRI contrast agents; Calcium sensitive; Lanthanide complexes; Luminescence
Synthesis and characterization of new copper thiosemicarbazone complexes with an ONNS quadridentate system: cell growth inhibition, S-phase cell cycle arrest and proapoptotic activities on cisplatin-resistant neuroblastoma cells by Haiyuan Zhang; Ronald Thomas; David Oupicky; Fangyu Peng (47-55).
Two new copper thiosemicarbazone complexes with an ONNS quadridentate system were synthesized and evaluated for anticancer activity on cisplatin-resistant neuroblastoma cells. Among these two copper complexes, the substituted 8-hydroxyquinoline-2-carboxaldehyde–4,4-dimethyl-3-thiosemicarbazide (CuHQDMTS) exhibited stronger cell growth inhibition activity than the unsubstituted copper 8-hydroxyquinoline-2-carboxaldehyde thiosemicarbazide complex (CuHQTS). Both CuHQTS and CuHQDMTS showed dose-dependent cell growth inhibition, cell cycle arrest and apoptosis induction activities on the SK-N-DZ neuroblastoma cells. Increased expression of p53 protein molecules was detected in the SK-N-DZ cells treated with CuHQTS. The data obtained in this study suggest that CuHQDMTS and CuHQTS hold potential as new, effective drugs for treatment of refractory neuroblastoma in children.
Keywords: Copper; Thiosemicarbazone; Proliferation inhibition; Cell cycle arrest; Apoptosis
Nature of non-transferrin-bound iron: studies on iron citrate complexes and thalassemic sera by Robert W. Evans; Roozina Rafique; Adel Zarea; Chiara Rapisarda; Richard Cammack; Patricia J. Evans; John B. Porter; Robert C. Hider (57-74).
Despite its importance in iron-overload diseases, little is known about the composition of plasma non-transferrin-bound iron (NTBI). Using 30-kDa ultrafiltration, plasma from thalassemic patients consisted of both filterable and non-filterable NTBI, the filterable fraction representing less than 10% NTBI. Low filterability could result from protein binding or NTBI species exceeding 30 kDa. The properties of iron citrate and its interaction with albumin were therefore investigated, as these represent likely NTBI species. Iron permeated 5- or 12-kDa ultrafiltration units completely when complexes were freshly prepared and citrate exceeded iron by tenfold, whereas with 30-kDa ultrafiltration units, permeation approached 100% at all molar ratios. A g = 4.3 electron paramagnetic resonance signal, characteristic of mononuclear iron, was detectable only with iron-to-citrate ratios above 1:100. The ability of both desferrioxamine and 1,2-dimethyl-3-hydroxypyridin-4-one to chelate iron in iron citrate complexes also increased with increasing ratios of citrate to iron. Incremental molar excesses of citrate thus favour the progressive appearance of chelatable lower molecular weight iron oligomers, dimers and ultimately monomers. Filtration of iron citrate in the presence of albumin showed substantial binding to albumin across a wide range of iron-to-citrate ratios and also increased accessibility of iron to chelators, reflecting a shift towards smaller oligomeric species. However, in vitro experiments using immunodepletion or absorption of albumin to Cibacron blue–Sepharose indicate that iron is only loosely bound in iron citrate–albumin complexes and that NTBI is unlikely to be albumin-bound to any significant extent in thalassemic sera.
Keywords: Albumin; Iron chelators; Iron citrate; Non-transferrin-bound iron; Thalassemia
A steady-state and pre-steady-state kinetics study of the tungstoenzyme formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus by Emile Bol; Nicolette J. Broers; Wilfred R. Hagen (75-84).
Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one [4Fe–4S] cubane per 69-kDa subunit. The enzyme kinetics have been studied under steady-state conditions at 80 °C and pre-steady state conditions at 50 °C, in the latter case via monitoring of the relatively weak (ε ≈ 2 mM−1 cm−1) optical spectrum of the tungsten cofactor. The steady-state data are consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The K M value for free formaldehyde (21 μM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor. The K M of ferredoxin (14 μM) is an order of magnitude less than previously reported values. An explanation for this discrepancy may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme. Pre-steady-state difference spectra reveal peak shifts and a lack of isosbestic points, an indication that several processes happen in the first seconds of the reaction. Two fast processes (k obs1 = 4.7 s−1, k obs2 = 1.9 s−1) are interpreted as oxidation of the substrate followed by rearrangement of the active site. Alternatively, these processes could be the entry/binding of the substrate followed by its oxidation. The release of the product and the electron shuffling over the tungsten and iron–sulfur center in the absence of an external electron acceptor are slower (k obs3 = 6.10 × 10−2 s−1, k obs4 = 2.18 × 10−2 s−1). On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation route plus catalytic redox cycle is proposed.
Keywords: Tungsten; Formaldehyde oxidoreductase; Pyrococcus furiosus ; Pre-steady-state kinetics; Steady-state kinetics
Active-site structure, binding and redox activity of the heme–thiolate enzyme CYP2D6 immobilized on coated Ag electrodes: a surface-enhanced resonance Raman scattering study by Alois Bonifacio; Diego Millo; Peter H. J. Keizers; Roald Boegschoten; Jan N. M. Commandeur; Nico P. E. Vermeulen; Cees Gooijer; Gert van der Zwan (85-96).
Surface-enhance resonance Raman scattering spectra of the heme–thiolate enzyme cytochrome P450 2D6 (CYP2D6) adsorbed on Ag electrodes coated with 11-mercaptoundecanoic acid (MUA) were obtained in various experimental conditions. An analysis of these spectra, and a comparison between them and the RR spectra of CYP2D6 in solution, indicated that the enzyme’s active site retained its nature of six-coordinated low-spin heme upon immobilization. Moreover, the spectral changes detected in the presence of dextromethorphan (a CYP2D6 substrate) and imidazole (an exogenous heme axial ligand) indicated that the immobilized enzyme also preserved its ability to reversibly bind a substrate and form a heme–imidazole complex. The reversibility of these processes could be easily verified by flowing alternately solutions of the various compounds and the buffer through a home-built spectroelectrochemical flow cell which contained a sample of immobilized protein, without the need to disassemble the cell between consecutive spectral data acquisitions. Despite immobilized CYP2D6 being effectively reduced by a sodium dithionite solution, electrochemical reduction via the Ag electrode was not able to completely reduce the enzyme, and led to its extensive inactivation. This behavior indicated that although the enzyme’s ability to exchange electrons is not altered by immobilization per se, MUA-coated electrodes are not suited to perform direct electrochemistry of CYP2D6.
Keywords: Surface-enhanced resonance Raman scattering; Cytochrome P450 2D6; Enzyme immobilization; Ag electrodes
The exchange activities of [Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) from methanogenic archaea in comparison with the exchange activities of [FeFe] and [NiFe] hydrogenases by Sonja Vogt; Erica J. Lyon; Seigo Shima; Rudolf K. Thauer (97-106).
[Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) catalyzes the reversible reduction of methenyltetrahydromethanopterin (methenyl-H4MPT+) with H2 to methylene-H4MPT, a reaction involved in methanogenesis from H2 and CO2 in many methanogenic archaea. The enzyme harbors an iron-containing cofactor, in which a low-spin iron is complexed by a pyridone, two CO and a cysteine sulfur. [Fe] hydrogenase is thus similar to [NiFe] and [FeFe] hydrogenases, in which a low-spin iron carbonyl complex, albeit in a dinuclear metal center, is also involved in H2 activation. Like the [NiFe] and [FeFe] hydrogenases, [Fe] hydrogenase catalyzes an active exchange of H2 with protons of water; however, this activity is dependent on the presence of the hydride-accepting methenyl-H4MPT+. In its absence the exchange activity is only 0.01% of that in its presence. The residual activity has been attributed to the presence of traces of methenyl-H4MPT+ in the enzyme preparations, but it could also reflect a weak binding of H2 to the iron in the absence of methenyl-H4MPT+. To test this we reinvestigated the exchange activity with [Fe] hydrogenase reconstituted from apoprotein heterologously produced in Escherichia coli and highly purified iron-containing cofactor and found that in the absence of added methenyl-H4MPT+ the exchange activity was below the detection limit of the tritium method employed (0.1 nmol min−1 mg−1). The finding reiterates that for H2 activation by [Fe] hydrogenase the presence of the hydride-accepting methenyl-H4MPT+ is essentially required. This differentiates [Fe] hydrogenase from [FeFe] and [NiFe] hydrogenases, which actively catalyze H2/H2O exchange in the absence of exogenous electron acceptors.
Keywords: Hydrogenase; Exchange reactions; Methanogenic archaea
Tuning the antiproliferative activity of biologically active iron chelators: characterization of the coordination chemistry and biological efficacy of 2-acetylpyridine and 2-benzoylpyridine hydrazone ligands by Paul V. Bernhardt; Gregory J. Wilson; Philip C. Sharpe; Danuta S. Kalinowski; Des R. Richardson (107-119).
2-Pyridinecarbaldehyde isonicotinoyl hydrazone (HPCIH) and di-2-pyridylketone isonicotinoyl hydrazone (HPKIH) are two Fe chelators with contrasting biological behavior. HPCIH is a well-tolerated Fe chelator with limited antiproliferative activity that has potential applications in the treatment of Fe-overload disease. In contrast, the structurally related HPKIH ligand possesses significant antiproliferative activity against cancer cells. The current work has focused on understanding the mechanisms of the Fe mobilization and antiproliferative activity of these hydrazone chelators by synthesizing new analogs (based on 2-acetylpyridine and 2-benzoylpyridine) that resemble both series and examining their Fe coordination and redox chemistry. The Fe mobilization activity of these compounds is strongly dependent on the hydrophobicity and solution isomeric form of the hydrazone (E or Z). Also, the antiproliferative activity of the hydrazone ligands was shown to be influenced by the redox properties of the Fe complexes. This indicated that toxic Fenton-derived free radicals are important for the antiproliferative activity for some hydrazone chelators. In fact, we show that any substitution of the H atom present at the imine C atom of the parent HPCIH analogs leads to an increase in antiproliferative efficacy owing to an increase in redox activity. These substituents may deactivate the imine R–C=N–Fe (R is Me, Ph, pyridyl) bond relative to when a H atom is present at this position preventing nucleophilic attack of hydroxide anion, leading to a reversible redox couple. This investigation describes novel structure–activity relationships of aroylhydrazone chelators that will be useful in designing new ligands or fine-tuning the activity of others.
Keywords: Iron chelator; Hydrazone; Cancer
Noninnocent effect of axial ligand on the heme degradation process: a theoretical approach to hydrolysis pathway of verdoheme to biliverdin by Parisa R. Jamaat; Nasser Safari; Mina Ghiasi; S. Shahab-al-din Naghavi; Mansour Zahedi (121-132).
Conversion of iron(II) verdoheme to iron(II) biliverdin in the presence of hydroxyl ion as a nucleophile and imidazole, pyridine, water, hydroxyl, cyanide, phenolate, chloride, thiolate and imidazolate as axial ligands was investigated using the B3LYP method and the 6-31G basis set. In the five-coordinated pathway the reactants and products are in the ground triplet state. In this path, hydroxyl ion directly attacks the macrocycle. The exothermic energy for addition of hydroxyl ion to iron(II) verdoheme with various ligands is 169.55, 166.34 and 164 kcal mol−1 for water, pyridine and imidazole, energies which are around 30–60 kcal mol−1 more exothermic than those for the other axial ligands used in this study. Therefore, imidazole, water and pyridine axial ligands can facilitate hydrolytic cleavage of iron(II) verdoheme to form open-chained helical iron(II) biliverdin complexes. The activation barrier for the conversion of iron(II) verdoheme hydroxyl species to the iron(II) biliverdin complex is estimated to be 5.2, 4.2, 4.35, 13.76 and 14.05 kcal mol−1 for imidazole, water, cyanide, thiolate and imidazolate, respectively.
Keywords: Verdoheme; Biliverdin; Density functional theory calculation; Hydrolysis; Hydroxyl ion
A theoretical study of zinc(II) interactions with amino acid models and peptide fragments by Bartosz Trzaskowski; Ludwik Adamowicz; Pierre A. Deymier (133-137).
Density functional theory calculations have been employed to study the interaction between the Zn2+ ion and some standard amino acid models. The highest affinities towards the Zn2+ ion are predicted for serine, cysteine, and histidine. Relatively high affinities are reported also for proline and glutamate/aspartate residues. It was found that the zinc complexes with cysteine adopt a tetrahedral conformation. Conversely, complexes with one or two histidine moieties remain in an octahedral geometry, while those with three or more histidine groups adopt a square-planar geometry.
Keywords: Density functional theory; Zinc; Amino acids; Metallization; Enzymes
Probing the role of the divalent metal ion in uteroferrin using metal ion replacement and a comparison to isostructural biomimetics by Gerhard Schenk; Rosely A. Peralta; Suzana Cimara Batista; Adailton J. Bortoluzzi; Bruno Szpoganicz; Andrew K. Dick; Paul Herrald; Graeme R. Hanson; Robert K. Szilagyi; Mark J. Riley; Lawrence R. Gahan; Ademir Neves (139-155).
Purple acid phosphatases (PAPs) are a group of heterovalent binuclear metalloenzymes that catalyze the hydrolysis of phosphomonoesters at acidic to neutral pH. While the metal ions are essential for catalysis, their precise roles are not fully understood. Here, the Fe(III)Ni(II) derivative of pig PAP (uteroferrin) was generated and its properties were compared with those of the native Fe(III)Fe(II) enzyme. The k cat of the Fe(III)Ni(II) derivative (approximately 60 s−1) is approximately 20% of that of native uteroferrin, and the Ni(II) uptake is considerably faster than the reconstitution of full enzymatic activity, suggesting a slow conformational change is required to attain optimal reactivity. An analysis of the pH dependence of the catalytic properties of Fe(III)Ni(II) uteroferrin indicates that the μ-hydroxide is the likely nucleophile. Thus, the Ni(II) derivative employs a mechanism similar to that proposed for the Ga(III)Zn(II) derivative of uteroferrin, but different from that of the native enzyme, which uses a terminal Fe(III)-bound nucleophile to initiate catalysis. Binuclear Fe(III)Ni(II) biomimetics with coordination environments similar to the coordination environment of uteroferrin were generated to provide both experimental benchmarks (structural and spectroscopic) and further insight into the catalytic mechanism of hydrolysis. The data are consistent with a reaction mechanism employing an Fe(III)-bound terminal hydroxide as a nucleophile, similar to that proposed for native uteroferrin and various related isostructural biomimetics. Thus, only in the uteroferrin-catalyzed reaction are the precise details of the catalytic mechanism sensitive to the metal ion composition, illustrating the significance of the dynamic ligand environment in the protein active site for the optimization of the catalytic efficiency.
Keywords: Binuclear metallohydrolases; Purple acid phosphatases; Uteroferrin; Catalysis; Metal ion replacement