JBIC Journal of Biological Inorganic Chemistry (v.7, #3)

The systems VIVO2+-glutathione and related ligands: a potentiometric and spectroscopic study by João Pessoa; Isabel Tomaz; Tamás Kiss; Erzsébet Kiss; Péter Buglyó (225-240).
The equilibria in the system VIVO2+-glutathione in aqueous solution were studied in the pH range 2–11 by a combination of pH-potentiometry and spectroscopy (EPR, visible absorption and circular dichroism). The results of the various methods are consistent and the equilibrium model includes the species MLH3, MLH2, MLH, ML2H2, MLH–1, and MLH–2 and several hydrolysis products (where H4L denotes totally protonated glutathione); individual formation constants and spectra are given. ML2H2 is the predominant species at physiological pH. Plausible structures for each stoichiometry are discussed. The related VIVO2+ systems of S-methylglutathione and γ-L-glutamyl-L-cysteinyl ethyl ester were studied by means of the same spectroscopic techniques in order to support the established binding modes for the glutathione complexes. The importance of glutathione and oxidized glutathione in binding VIVO2+ in cells is assessed.
Keywords: Vanadium Glutathione Potentiometry Spectroscopy Coordination modes

Stability and transmetallation of the magnetic resonance contrast agent MnDPDP measured by EPR by Peter P. Schmidt; Kim G. Toft; Tore Skotland; Kristoffer K. Andersson (241-248).
MnDPDP [manganese(II) N,N′-dipyridoxylethylenediamine-N,N′-diacetate-5,5′-bis(phosphate)] is the active component of Teslascan, a contrast medium for magnetic resonance imaging of the liver. It has previously been shown that MnDPDP is rapidly dephosphorylated to the monophosphate MnDPMP and the non-phosphorylated MnPLED, and that all these substances are rapidly transmetallated to the corresponding Zn complexes. In the present study we used EPR at 9 and 230 GHz to show that no free Mn2+ ions can be detected in the product or in a mixture of MnDPDP and human serum. Competition experiments between MnDPDP and Zn2+, Ca2+, and Mg2+ ions revealed approximately 15% transmetallation with Zn2+ in a buffer system containing metal ion concentrations similar to that in serum, whereas approximately 10% transmetallation was obtained with Ca2+ and only negligible transmetallation was obtained with Mg2+ under these conditions. Binding experiments with Mn2+ added to human albumin and human serum indicate that albumin accounts for most of the protein-bound Mn2+ in serum.
Keywords: MnDPDP EPR Transmetallation Protein binding Contrast agent

Copper bis(thiosemicarbazone) complexes as hypoxia imaging agents: structure-activity relationships by Jason L. Dearling; Jason S. Lewis; Gregory E. Mullen; Michael J. Welch; Philip J. Blower (249-259).
Copper(II) bis(thiosemicarbazone) complexes labelled with Cu-60/62/64 are useful radiopharmaceuticals for imaging blood flow and hypoxic tissues in vivo. The aim of this study was to identify structure-activity relationships within a series of analogues with different alkyl substitution patterns in the ligand, in order to design improved hypoxia imaging agents and elucidate hypoxia selectivity mechanisms. Thirteen such complexes were synthesised and characterised spectroscopically and electrochemically. The uptake of each (labelled with Cu-64) in EMT6 tumour cells in vitro under normoxic and hypoxic conditions was studied. All complexes were taken up efficiently into cells, and some showed strong hypoxia selectivity, which was highly correlated with the Cu(II/I) redox potential. Redox potentials at the low end of the range were found to be essential for hypoxia selectivity. In turn, the redox potential was strongly correlated with alkyl substitution pattern, and the most important determinant of the redox potential was the number of alkyl groups on the diimine backbone of the ligand. Several complexes in the series warrant further evaluation as hypoxia imaging agents. The radioactivity uptake/release behaviour in the cells provides insight into possible mechanisms, and a model for hypoxia-selective intracellular trapping is discussed.
Keywords: Radiopharmaceutical Imaging agents Copper bis(thiosemicarbazone) Hypoxia Redox potential

Hydrogenobacter thermophilus cytochrome c 552 (Ht cyt c 552) is a small monoheme protein in the cytochrome c 551 family. Ht cyt c 552 is unique because it is hypothesized to undergo spontaneous cytoplasmic maturation (covalent heme attachment) when expressed in Escherichia coli. This is in contrast to the usual maturation route for bacterial cytochromes c that occurs in the cellular periplasm, where maturation factors direct heme attachment. Here, the expression of Ht cyts c 552 in the periplasm as well as the cytoplasm of E. coli is reported. The products are characterized by absorption, circular dichroism, and NMR spectroscopy as well as mass spectrometry, proteolysis, and denaturation studies. The periplasmic product's properties are found to be indistinguishable from those reported for protein isolated from Ht cells, while the major cytoplasmic product exhibits structural anomalies in the region of the N-terminal helix. These anomalies are shown to result from the retention of the N-terminal methionine in the cytoplasmic product, and not from heme attachment errors. The 1H NMR chemical shifts of the heme methyls of the oxidized (S=1/2) expression products display a unique pattern not previously reported for a cytochrome c with histidine-methionine axial ligation, although they are consistent with native-like heme ligation. These results support the hypothesis that proper heme attachment can occur spontaneously in the E. coli cytoplasm for Ht cyt c 552.
Keywords: Cytochrome c maturation Hydrogenobacter thermophilus Protein expression NMR spectroscopy Paramagnetism

The nature of the heme environment in methemalbumin, the Fe(III) protoporphyrin IX (heme)-human serum albumin (HSA) complex, was investigated by optical spectroscopy. Comparison of the optical spectra of methemalbumin, ferro-hemalbumin in the absence and presence of 2-methylimidazole, and their carbon monoxide derivatives with horseradish peroxidase (HRP) and its corresponding derivatives indicates that histidine is not present in the first coordination sphere of heme in methemalbumin and that the protein is devoid of a well-defined heme cavity. The complex exhibits peroxidase activity by catalyzing oxidation of 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) by hydrogen peroxide. Its activity (K M=433 µM, molar catalytic activity=0.33 s–1), however, is considerably lower compared to HRP, indicating differences in the heme environments. Fluorescence intensity decays of Trp214 in HSA and methemalbumin, best fitted to a three-exponential model, gave the lifetimes 7.03 ns (30%), 3.17 ns (38%), and 0.68 ns (32%) for HSA and 8.04 ns (1.7%), 2.42 ns (19.7%), and 0.64 ns (78.6%) for methemalbumin. These lifetime values were further confirmed by a model-independent maximum entropy method. Similarity in the lifetimes and variations in the amplitudes suggest that while conformational heterogeneity of HSA is unperturbed on heme binding, redistribution of the populations of the three conformations occurs and the sub-state associated with the shortest lifetime dominates the total population by ~80%. Decay associated spectra (DAS) indicate that the observed lifetime variation with wavelength is predominantly due to ground state heterogeneity, though solvent dipolar relaxation also contributes. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information on motion within the protein together with the whole protein molecule. The binding of heme did not affect the rotational correlation time of the albumin molecule (~20 ns). However, the motion of tryptophan within the protein matrix increased by a factor of ~3 (0.46 ns to 0.15 ns). This indicates that while the overall hydrodynamic volume of the albumin molecule remained the same, tryptophan underwent a more rapid internal rotation because of the efficient energy transfer to the bound heme. Optical studies, analysis of lifetime measurements, DAS, and anisotropy measurements together suggest that heme binds to a surface residue. The rapid internal motion of Trp214 during its excited state lifetime for the ~80% populated conformer of methemalbumin allows the orientation factor, κ2, to approach the average value of 2/3. From the time-resolved fluorescence measurements and the energy transfer calculations on methemalbumin, a Trp214-heme distance of 22 Å was deduced.
Keywords: Human serum albumin Methemalbumin ABTS oxidation Time-resolved fluorescence Decay associated spectra

A theoretical study of the mechanism for peptide hydrolysis by thermolysin by Vladimir Pelmenschikov; Margareta R. Blomberg; Per E. Siegbahn (284-298).
The catalytic mechanism for peptide hydrolysis by thermolysin has been investigated using the B3LYP hybrid density functional method. The starting structure for the calculations was based on the X-ray crystal structure of the enzyme inhibited with the ZF p LA phosphonamidate transition-state analogue. Besides the three Zn ligands His142, His146 and Glu166, a few additional residues were also included in the model. Following the order of importance, the outer-sphere ligands Glu143, His231 and Asp226 were shown to play significant catalytic roles, well correlated with results from site-directed mutagenesis experiments. A single-step reaction mechanism was obtained starting from the initial enzyme-substrate complex with a pentacoordinated metal center and proceeding to the enzyme-carboxylate complex as a final product, following a proposal by Matthews and co-workers. The transition state combines a nucleophilic water oxygen attack on the peptide carbon and a proton transfer from the water to the peptide nitrogen, mediated by the Glu143 carboxylate. A free activation energy of 15.2 kcal/mol was obtained, compared to the experimental 12.4–16.3 kcal/mol range for various peptide substrates. An interesting aspect of the present single-step mechanism is that the Glu143 carboxylate moves a significant distance of ~1.0 Å. Different chemical models were examined, both related to the system size and proper side-chain modeling. The significance of the protein frame rigidity around the active site was estimated by fixing and subsequently releasing the edge atom positions. Finally, alternative mechanistic proposals are briefly summarized.
Keywords: Thermolysin Peptide hydrolysis Catalytic mechanism Density functional theory

The biological conversions of O2 and peroxides to water as well as certain incorporations of oxygen atoms into small organic molecules can be catalyzed by metal ions in different clusters or cofactors. The catalytic cycle of these reactions passes through similar metal-based complexes in which one oxygen- or peroxide-derived oxygen atom is coordinated to an oxidized form of the catalytic metal center. In haem-based peroxidases or oxygenases the ferryl (FeIVO) form is important in compound I and compound II, which are two and one oxidation equivalents higher than the ferric (FeIII) form, respectively. In this study we report the 1.35 Å structure of a compound II model protein, obtained by reacting hydrogen peroxide with ferric myoglobin at pH 5.2. The molecular geometry is virtually unchanged compared to the ferric form, indicating that these reactive intermediates do not undergo large structural changes. It is further suggested that at low pH the dominating compound II resonance form is a hydroxyl radical ferric iron rather than an oxo-ferryl form, based on the short hydrogen bonding to the distal histidine (2.70 Å) and the Fe···O distance. The 1.92 Å Fe···O distance is in agreement with an EXAFS study of compound II in horseradish peroxidase.
Keywords: Myoglobin Compound II Haem Hydroxyl radical Oxygen activation

The molecular modeling of Co(II)-bleomycin previously performed by us through NMR and molecular dynamics indicates that the most favorable structure for this complex is six-coordinate, with the secondary amine in β-aminoalanine, the N5 and N1 nitrogens in the pyrimidine and imidazole rings, respectively, and the amide nitrogen in β-hydroxyhistidine as equatorial ligands. The primary amine and either the carbamoyl group or a solvent molecule are proposed to occupy the axial sites. In this report, the results of the molecular modeling of Fe(II)-bleomycin are presented. The NMR data for the ferrous derivative of the drug have already been reported by us, and were used here to generate the necessary restraints for this modeling work. For Co(II)-bleomycin, two new models exhibiting N-carbamoyl ligation to the metal centers were also assayed and compared with the ones previously examined. The results of this investigation on Fe(II)- and Co(II)-bleomycin are most consistent with a six-coordinate structure with five endogenous N-donors and a solvent molecule or the carbamoyl group as the sixth ligand. Comparisons of the best Co(II)- and Fe(II)-bleomycin models with the NMR-generated structures for some relevant metallo-BLMs favor the model with only endogenous ligands and N-carbamoyl ligation as the structure probably held in solution by both Co(II)- and Fe(II)-bleomycin.
Keywords: Bleomycin Iron Nuclear magnetic resonance Molecular modeling Structure

Interactions in solution of cobalt(II) and nickel(II) with nicotinamide adenine dinucleotide: a potentiometric and calorimetric study by Luis A. Herrero; José Cerro-Garrido; María Apella; Angel Terrón-Homar (313-317).
The equilibrium constants and the thermodynamic parameters enthalpy and entropy of the interaction between Ni(II) and Co(II) with NAD+ in aqueous solution were determined by calorimetry and potentiometry methods (ionic strength adjusted to 0.1 with sodium nitrate at 25 °C). The macrochelation of the systems was also studied. All the data, including the protonation enthalpy data of NAD+ (very similar to the protonation enthalpy of 5′-AMP) suggest a less restrictive model for the NAD+ "folded" conformation without intramolecular stacking between the bases, in agreement both with recent theoretical calculations and with the X-ray structure of trimethylene-bisadeninium or the free acid form of NAD+.
Keywords: Cobalt Nickel Nucleotides Nicotinamide adenine dinucleotide calorimetry

IR spectroelectrochemical study of the binding of carbon monoxide to the active site of Desulfovibrio fructosovorans Ni-Fe hydrogenase by Antonio L. De Lacey; Christian Stadler; Victor M. Fernandez; Claude E. Hatchikian; Hua-Jun Fan; Shuhua Li; Michael B. Hall (318-326).
The binding of carbon monoxide, a competitive inhibitor of many hydrogenases, to the active site of Desulfovibrio fructosovorans hydrogenase has been studied by infrared spectroscopy in a spectroelectrochemical cell. Direct evidence has been obtained of which redox states of the enzyme can bind extrinsic CO. Redox states A, B and SU do not bind extrinsic CO; only after reductive activation of the hydrogenase can CO bind to the active site. Two states with bound extrinsic CO can be distinguished by FTIR. These two states are in redox equilibrium and are most probably due to different oxidation states of the proximal 4Fe-4S cluster. Vibrational frequencies and theoretical quantum mechanics studies (DFT) of this process preclude the possibility of strong bonding of extrinsic CO to the Fe or Ni atoms of the active site. We propose that CO inhibition is caused by weak interaction of the extrinsic ligand with the Ni atom, blocking electron and proton transfer at the active site. A calculated structure with a weakly bound extrinsic CO at Ni has relative CO frequencies in excellent agreement with the experimental ones.
Keywords: Metalloprotein Fourier transform IR Spectroelectrochemistry Carbon monoxide Density functional theory

The thermodynamics of Cu(II) and Ni(II) binding to bovine serum albumin (BSA) have been studied by isothermal titration calorimetry (ITC). The Cu(II) binding affinity of the N-terminal protein site is quantitatively higher when the single free thiol, Cys-34, is reduced (mercaptalbumin), compared to when it is oxidized or derivatized with N-ethylmaleimide. This increased affinity is due predominantly to entropic factors. At higher pH (~9), when the protein is in the basic (B) form, a second Cu(II) binds with high affinity to albumin with reduced Cys-34. The Cu(II) coordination has been characterized by UV-vis absorption, CD, and EPR spectroscopy, and the spectral data are consistent with thiolate coordination to a tetragonal Cu(II), indicating this is a type 2 copper site with thiolate ligation. Nickel(II) binding to the N-terminal site of BSA is also modulated by the redox/ligation state of Cys-34, with higher Ni(II) affinity for mercaptalbumin, the predominant circulating form of the protein.
Keywords: Bovine serum albumin Isothermal titration calorimetry Copper(II) binding Nickel(II) binding

Pulsed ELDOR spectroscopy of the Mo(V)/Fe(III) state of sulfite oxidase prepared by one-electron reduction with Ti(III) citrate by Rachel Codd; Andrei V. Astashkin; Andrew Pacheco; Arnold M. Raitsimring; John H. Enemark (338-350).
The titration of chicken liver sulfite oxidase (SO) with the one-electron reductant Ti(III) citrate, at pH 7.0, results in nearly quantitative selective reduction of the Mo(VI) center to Mo(V), while the b-type heme center remains in the fully oxidized Fe(III) state. The selective reduction of the Mo(VI/V) couple has been established from electronic and EPR spectra. The electronic spectrum of the Fe(III) heme center is essentially unchanged during the titration, and the continuous wave (CW)-EPR spectrum shows the appearance of the well-known Mo(V) signal due to the low pH (lpH) form of SO. Further confirmation of the selective formation of the Mo(V)/Fe(III) form of SO is provided by the ~1:1 ratio of the integrated intensities of the Mo(V) and low-spin Fe(III) EPR signals after addition of one equivalent of Ti(III). The selective generation of the Mo(V)/Fe(III) form of SO is unexpected, considering that previous microcoulometry and flash photolysis investigations have indicated that the Mo(VI/V) and Fe(III/II) couples of SO have similar reduction potentials at pH 7. The nearly quantitative preparation of the one-electron reduced Mo(V)/Fe(III) form of SO by reduction with Ti(III) has enabled the interaction between these two paramagnetic metal centers, which are linked by a flexible loop with no secondary structure, to be investigated for the first time by variable-frequency pulsed electron-electron double resonance (ELDOR) spectroscopy. The ELDOR kinetics were obtained from frozen solutions at 4.2 K at several microwave frequencies by pumping on the narrow Mo(V) signal and observing the effect on the Fe(III) primary echo at both higher and lower frequencies within the microwave C-band region. The ELDOR data indicate that freezing the solution of one-electron reduced SO produces localized regions where the concentration of SO approaches that in the crystal structure, which results in the interpair interactions being the dominant dipolar interaction. However, thorough analysis of the ELDOR decay curves and simulations suggests a distribution of intramolecular Mo...Fe distances, consistent with the proposal of multiple conformations in solution for the flexible loop that connects the Mo and heme domains of SO.
Keywords: Sulfite oxidase Molybdenum-containing enzyme Electron-electron double resonance Titanium(III) citrate Intramolecular electron transfer

Erratum to Oxidative chemistry of nickel porphyrins by Mark W. Renner; Jack Fajer (352-352).

Meeting of Interest (353-353).