JBIC Journal of Biological Inorganic Chemistry (v.7, #1-2)

Editorial by Larry Que (1-1).

Four of the five intermediate oxidation states (S-states) in the catalytic cycle of water oxidation used by O2-evolving photoautotrophs have been previously characterized by EPR and/or ENDOR spectroscopy, with the first reports for the S0, S1, and S3 states available in just the last three years. The first electron density map of the Mn cluster derived from X-ray diffraction measurements of single crystals of photosystem II at 3.8–4.2 Å resolution has also appeared this year. This wealth of new information has provided significant insight into the structure of the inorganic core (Mn4O x Ca1Cl1–2), the Mn oxidation states, and the location and function of the essential Ca2+ cofactor within the water-oxidizing complex (WOC). We summarize these advances and provide a unified interpretation of debated structural proposals and Mn oxidation states, based on an integrated analysis of the published data, particularly from Mn X-ray absorption spectroscopy (XAS) and EPR/ENDOR data. Only three magnetic spin-exchange models for the inter-manganese interactions are possible from consideration of the EPR data for the S0, S1, S2 and S N (NO-reduced) states. These models fall into one of three types denoted butterfly, funnel, or tetrahedron. A revised set of eight allowed chemical structures for the Mn4O x core can be deduced that are shown to be consistent with both EPR and XAS. The popular "dimer-of-dimers" structural model is not compatible with the possible structural candidates. EPR data have identified two inter-manganese couplings that are sensitive to the S-state, suggesting two possible bridging sites for substrate water molecules. Spin densities derived from 55Mn hyperfine data together with Mn K-edge energies from Ca-depleted samples provide an internally consistent assignment for the Mn oxidation states of Mn4(3III,IV) for the S2 state. EPR and XAS data also provide a consistent picture, locating Ca2+ as an integral part of the inorganic core, probably via shared bridging ligands with Mn (aqua/hydroxo/carboxylato/chloro). XAS data reveal that the Ca2+ cofactor increases the Mn(1s→4p) transition energy by 0.6–1 eV with minimal structural perturbation versus the Ca-depleted WOC. Thus, calcium binding appears to increase the Mn-ligand covalency by increasing electron transfer from shared ligands to Mn, suggesting a direct role for Ca2+ in substrate water oxidation. Consideration of both the XAS and the EPR data, together with reactivity studies on two model complexes that evolve O2, suggest two favored structure types as feasible models for the reactive S4 state that is precursor to the O2 evolution step. These are a calcium-capped "cuboidal" core and a calcium-capped "funnel" core.
Keywords: Photosynthesis Catalysis Oxygen evolution Water oxidation Manganese

Using Brownian motion simulations we have studied the formation of docked complexes of reduced cytochrome b 5 and oxidized haemoglobin. Our results indicate that the presence of molecular electrostatic fields has a significant role to play in the formation of these complexes. In contrast to previous modeling studies on this system, we clearly identify electron transfer within an ensemble of similarly docked complexes rather than the formation of a single complex. Docking involves a number of acidic residues surrounding the exposed haem edge of cytochrome b 5 and a set of basic residues surrounding the exposed haem edge of the globins. Although amino acids from the partner globin proteins are involved to a small extent in the binding of some of the complexes, the reactivity of any particular globin is essentially independent of the nature of its partner globin chain within the haemoglobin molecule. Comparison of results from adult and embryonic haemoglobins indicates a significant difference in complex formation. Application of electron tunneling analysis to the complexes allows us to predict the rates of electron transfer within each ensemble of complexes. These data provide a theoretical insight into the important process of re-reduction of oxidized haemoglobins as well as explaining the general inability to produce crystalline forms of many docked electron transfer complexes.
Keywords: Cytochrome b5 Haemoglobin Brownian simulation Electron transfer

CO2 catalyses the isomerization of the biological toxin ONOO to NO3 via an intermediate, presumably ONOOCO2 , which has an absorption maximum near 650 nm. The reflection spectrum of solid NMe4 +ONOO exposed to CO2 shows a similar band near 650 nm; this absorption decays over minutes. Stopped-flow experiments in which CO2 solutions were mixed with alkaline ONOO solutions indicate the formation of at least one intermediate. The initial absorption at 302 nm is less than that of ONOO, which indicates that reactions take place within the mixing time, and this absorption is dependent (but not linearly) on the ONOO and CO2 concentrations. We found that reaction of peroxynitrite with carbon dioxide forms some trioxocarbonate(•1–) (CO3 •–) and nitrogen dioxide (NO2 ) radicals via homolysis of the O-O bond in ONOOCO2 . We determined the extent of radical formation by mixing peroxynitrite, carbon dioxide and nitrogen monoxide. The later reacts with CO3 •– and NO2 radicals to form, effectively, three NO2 per homolysis; ONOOCO2 that does not undergo homolysis yields NO3 and CO2. Based on the NO3 and NO2 analyses, the extent of conversion to NO3 is 96±1% and that of homolysis is 3±1%, respectively, significantly less than that reported in the literature.
Keywords: Peroxynitrite Carbon dioxide Trioxocarbonate(•1–) Stopped-flow spectrophotometry

Samples of the dithionite-reduced FeFe protein (the dinitrogenase component of the Fe-only nitrogenase) from Rhodobacter capsulatus have been investigated by 57Fe Mössbauer spectroscopy and by Fe and Zn EXAFS as well as XANES spectroscopy. The analyses were performed on the basis of data known for the FeMo cofactor and the P cluster of Mo nitrogenases. The prominent Fourier transform peaks of the Fe K-edge spectrum are assigned to Fe-S and Fe-Fe interactions at distances of 2.29 Å and 2.63 Å, respectively. A significant contribution to the Fe EXAFS must be assigned to an Fe backscatterer shell at 3.68 Å, which is an unprecedented feature of the trigonal prismatic arrangement of iron atoms found in the FeMo cofactor of nitrogenase MoFe protein crystal structures. Additional Fe...Fe interactions at 2.92 Å and 4.05 Å clearly indicate that the principal geometry of the P cluster is also conserved. Mössbauer spectra of 57Fe-enriched FeFe protein preparations were recorded at 77 K (20 mT) and 4.2 K (20 mT, 6.2 T), whereby the 4.2 K high-field spectrum clearly demonstrates that the cofactor of the Fe-only nitrogenase (FeFe cofactor) is diamagnetic in the dithionite-reduced ("as isolated") state. The evaluation of the 77 K spectrum is in agreement with the assumption that this cofactor contains eight Fe atoms. In the literature, several genetic and biochemical lines of evidence are presented pointing to a significant structural similarity of the FeFe, the FeMo and and the FeV cofactors. The data reported here provide the first spectroscopic evidence for a structural homology of the FeFe cofactor to the heterometal-containing cofactors, thus substantiating that the FeFe cofactor is the largest iron-sulfur cluster so far found in nature.
Keywords: Rhodobacter capsulatus Fe nitrogenase FeFe cofactor Mössbauer spectroscopy Extended X-ray absorption fine structure

1H NMR investigations of the molecular nature of low-molecular-mass calcium ions in biofluids by Christopher J. Silwood; Martin Grootveld; Edward Lynch (46-57).
High-resolution 1H NMR spectroscopy was employed to explore the complexation of Ca2+ by low-molecular-mass biomolecules in human saliva. The results acquired revealed that the organic acid anion (OAA) citrate acts as a powerful oxygen-donor chelator for salivary Ca2+, and accurate determination of its resonances' frequencies and spin-system pattern could be successfully utilized to determine its degree of saturation with this metal ion. Computer modelling studies demonstrated that the OAA lactate is the only competing salivary Ca2+ complexant available. Moreover, the Ca2+-complexation status of salivary citrate is substantially modified by dentifrice-mediated elevations in its concentration. 1H NMR analysis was also applied to determinations of the Ca2+ saturation status of citrate in a variety of alternative biofluids and the biochemical significance of these results is discussed.
Keywords: Calcium citrate Speciation Biofluids Organic acid anion ligands 1H NMR spectroscopy

Relaxometric evaluation of novel manganese(II) complexes for application as contrast agents in magnetic resonance imaging by Silvio Aime; Pier Anelli; Mauro Botta; Marino Brocchetta; Simonetta Canton; Franco Fedeli; Eliana Gianolio; Enzo Terreno (58-67).
Three novel Mn(II) complexes bearing benzyloxymethyl functionalities are reported and their ability to enhance water (1H and 17O) relaxation times is investigated in detail. Two of them contain one coordinated water molecule and display relaxivity values only slightly smaller than those shown by the most clinically used contrast agents (e.g. [Gd(DTPA)(H2O)]2–). Moreover, in these Mn(II) chelates the exchange rate of the coordinated water is ca. one order of magnitude higher if compared to the exchange rates previously reported for Gd(III) complexes with octadentate ligands. The occurrence of such fast exchange rates of the coordinated water is exploited in the formation of macromolecular adducts with human serum albumin to attain systems displaying relaxivity values in the upper range of those so far reported for analogous Gd(III) systems. These results strongly support the view that Mn(II) complexes, in spite of the lower effective magnetic moment, can be considered as viable alternatives to the currently used Gd(III) complexes as contrast agents for MRI applications.
Keywords: Manganese(II) chelates Contrast agents Human serum albumin Water exchange rate Relaxometry

Development of new insulinomimetic zinc(II) picolinate complexes with a Zn(N2O2) coordination mode: structure characterization, in vitro, and in vivo studies by Yutaka Yoshikawa; Eriko Ueda; Kenji Kawabe; Hiroyuki Miyake; Toshikazu Takino; Hiromu Sakurai; Yoshitane Kojima (68-73).
Three zinc(II) complexes of picolinic acid and its derivatives with a Zn(N2O2) coordination mode were prepared and evaluated for their insulinomimetic activities by in vitro and in vivo studies. By introducing an electron-donating methyl group into the picolinate ligand (pic), bis(6- or 3-methylpicolinato)zinc(II) complexes [Zn(6-mpa)2 or Zn(3-mpa)2, respectively] were prepared. The Zn(6-mpa)2 complex was crystallized as a water adduct [Zn(6-mpa)2(H2O)]·H2O, in which two carboxylate oxygens and two pyridine nitrogens of 6-mpa and a water oxygen coordinate to a zinc(II) with a trigonal bipyramidal geometry. By in vitro evaluation of the inhibition of free fatty acid (FFA) release from isolated rat adipocytes in the presence of epinephrine, the insulinomimetic activities of Zn(pic)2, Zn(6-mpa)2, and Zn(3-mpa)2 (IC50=0.64±0.13, 0.31±0.05, and 0.40±0.07 mM, respectively) were found to be higher than those of VOSO4 (IC50=1.00 mM) and ZnSO4 (IC50=1.58±0.05 mM) in terms of IC50 value, the 50% inhibition concentrations for the FFA release from the adipocytes. Then, Zn(6-mpa)2, which exhibited the highest in vitro insulinomimetic activity among three complexes examined, was given at a dose of 3.0 mg (45.9 µmol) Zn/kg body weight to KK-Ay mice with type 2 diabetes mellitus by daily intraperitoneal injections for 14 days and it was found that the hereditary high blood glucose levels were lowered during the administration of the complex. The improvement of diabetes mellitus was confirmed with the oral glucose tolerance test.
Keywords: Zinc(II) complex Picolinic acid derivatives Insulinomimetic activity KK-Ay mice Blood glucose normalizing effect

Peroxyl adduct radicals formed in the iron/oxygen reconstitution reaction of mutant ribonucleotide reductase R2 proteins from Escherichia coli by Margareta Sahlin; Kyung-Bin Cho; Stephan Pötsch; Simon D. Lytton; Yasmin Huque; Michael R. Gunther; Britt-Marie Sjöberg; Ronald P. Mason; Astrid Gräslund (74-82).
Catalytically important free radicals in enzymes are generally formed at highly specific sites, but the specificity is often lost in point mutants where crucial residues have been changed. Among the transient free radicals earlier found in the Y122F mutant of protein R2 in Escherichia coli ribonucleotide reductase after reconstitution with Fe2+ and O2, two were identified as tryptophan radicals. A third radical has an axially symmetric EPR spectrum, and is shown here using 17O exchange and simulations of EPR spectra to be a peroxyl adduct radical. Reconstitution of other mutants of protein R2 (i.e. Y122F/W48Y and Y122F/W107Y) implicates W48 as the origin of the peroxyl adduct. The results indicate that peroxyl radicals form on primary transient radicals on surface residues such as W48, which is accessible to oxygen. However, the specificity of the reaction is not absolute since the single mutant W48Y also gives rise to a peroxyl adduct radical. We used density functional calculations to investigate residue-specific effects on hyperfine coupling constants using models of tryptophan, tyrosine, glycine and cysteine. The results indicate that any peroxyl adduct radical attached to the first three amino acid α-carbons gives similar 17O hyperfine coupling constants. Structural arguments and experimental results favor W48 as the major site of peroxyl adducts in the mutant Y122F. Available molecular oxygen can be considered as a spin trap for surface-located protein free radicals.
Keywords: Peroxyl radical Tryptophan radical Tyrosyl radical Ribonucleotide reductase Radical transfer pathways

The iron-sulfur center of biotin synthase: site-directed mutants by Kirsty S. Hewitson; Sandrine Ollagnier-de Choudens; Yiannis Sanakis; Nicholas M. Shaw; Jack E. Baldwin; Eckard Münck; Peter L. Roach; Marc Fontecave (83-93).
Biotin synthase contains an essential [4Fe-4S]+ cluster that is thought to provide an electron for the cleavage of S-adenosylmethionine, a cofactor required for biotin formation. The conserved cysteine residues Cys53, Cys57 and Cys60 have been proposed as ligands to the [4Fe-4S] cluster. These residues belong to a C-X3-C-X2-C motif which is also found in pyruvate formate lyase-activating enzyme, lysine 2,3-aminomutase and the anaerobic ribonucleotide reductase-activating component. To investigate the role of the cysteine residues, Cys→Ala mutants of the eight cysteine residues of Escherichia coli biotin synthase were prepared and assayed for activity. Our results show that six cysteines are important for biotin formation. Only two mutant proteins, C276A and C288A, closely resembled the wild-type protein, indicating that the corresponding cysteines are not involved in iron chelation and biotin formation. The six other mutant proteins, C53A, C57A, C60A, C97A, C128A and C188A, were inactive but capable of assembling a [4Fe-4S] cluster, as shown by Mössbauer spectroscopy. The C53A, C57A and C60A mutant proteins are unique in that their cluster could not undergo reduction to the [4Fe-4S]+ state, as shown by EPR and Mössbauer spectroscopy. On this basis and by analogy with pyruvate formate lyase-activating enzyme and the anaerobic ribonucleotide reductase-activating component, it is suggested that the corresponding cysteines coordinate the cluster even though one cannot fully exclude the possibility that other cysteines play that role as well. Therefore it appears that for activity biotin synthase absolutely requires cysteines that are not involved in iron chelation.
Keywords: Biotin synthase Iron-sulfur Mössbauer EPR Mutagenesis

The pH-dependent redox inactivation of amicyanin from Paracoccus versutus as studied by rapid protein-film voltammetry by Lars J. Jeuken; Raul Camba; Fraser A. Armstrong; Gerard W. Canters (94-100).
The redox properties of the blue copper protein amicyanin have been studied with slow and fast scan protein-film cyclic voltammetry. At slow scan rates, which reveal the thermodynamics of the redox reactions, the reduction potential of amicyanin depends on pH in a sigmoidal manner, and the data can be analysed in terms of electron transfer being coupled to a single protonatable group with pKa red=6.3 and pKa ox≤3.2 at 22 °C. Voltammetry at higher scan rates reveals the kinetics and shows that the low-pH reduced form of amicyanin is not oxidised directly; instead, oxidation occurs only after conversion to the high-pH form. Simulations show that this conversion, which gates the electron transfer, occurs with a rate constant >750 s–1 at 25 °C. In order to decrease the rate of the coupled reaction, the experiments were performed at 0 °C, at which the rate constant for this conversion was determined to be 35±20 s–1. Together with evidence from NMR, the results lead to a mechanism involving protonation and dissociation of the copper coordinating histidine-96 in the reduced form.
Keywords: Potentiometry Blue copper protein Histidine protonation Kinetics

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M and coenzyme B in methanogenic archaea. The enzyme contains tightly bound the nickel porphinoid F430. The nickel enzyme has been shown to be active only when its prosthetic group is in the Ni(I) reduced state. In this state MCR exhibits the nickel-based EPR signal red1. We report here for the MCR from Methanothermobacter marburgensis that the EPR spectrum of the active enzyme changed upon addition or removal of coenzyme M, methyl coenzyme M and/or coenzyme B. In the presence of methyl-coenzyme M the red1 signal showed a more resolved 14N-superhyperfine splitting than in the presence of coenzyme M indicating a possible axial ligation of the substrate to the Ni(I). In the presence of methyl-coenzyme M and coenzyme B the red1 signal was the same as in the presence of methyl-coenzyme M alone. However, in the presence of coenzyme M and coenzyme B a highly rhombic EPR signal, MCR-red2, was induced, which was found to be light sensitive and appeared to be formed at the expense of the MCR-red1 signal. Upon addition of methyl-coenzyme M, the red2 signal disappeared and the red1 signal increased again. The red2 signal of MCR with 61Ni-labeled cofactor was significantly broadened indicating that the signal is nickel or nickel-ligand based.
Keywords: Methyl-coenzyme M reductase Nickel enzymes Factor 430 Methanogenic archaea EPR spectroscopy

Stopped-flow kinetic study of the peroxidase reactions of mangano-microperoxidase-8 by Hui-Chun Yeh; Chia-Huei Yu; Jinn-Shyan Wang; Suei-Tein Chen; Oliver Y. Su; Wann-Yin Lin (113-119).
We have investigated the kinetics for the peroxidase-type reaction of mangano microperoxidase 8 (Mn(III)-MP8) by the time-resolved and single-wavelength stopped-flow technique. The formation of intermediate and its subsequent reaction with substrates were studied separately. Oxidation of Mn(III)-MP8 by H2O2 at pH 10.7 yields an intermediate (1) with a rate constant of 2.9×104 M–1 s–1. The formation of 1 exhibits no deuterium solvent isotope effect, favoring the homolytic cleavage of the Mn(III)-MP8 bound hydroperoxide. The rate for the formation of 1 increases sharply as the pH increases and no other intermediate was detected in the entire pH range. Addition of substrate to 1 leads to the regeneration of Mn(III)-MP8. Monitoring the conversion of 1 to Mn(III)-MP8 allows the determination of the substrate reactivity. The substrate reactivity varies by more than two orders of magnitude ranging from 1.04×106 M–1s–1 for ascorbic acid to 4.61×103 M–1s–1 for aniline. It is linearly correlated with the reduction potential for most of the substrates studied, with the easier oxidized species showing greater reactivity. The substrate reactivity drops rapidly as the pH increases. The substrate reactivity at pH 10.7 for the Mn(III)-MP8 system is smaller than that of the corresponding Fe(III)-MP8 system by 2- to 25-fold, depending on the substrate used.
Keywords: Mn(III)-microperoxidase 8 Kinetic study Substrate reactivity

Comparison of the X-ray absorption properties of the binuclear active site of molluscan and arthropodan hemocyanins by A. Sabatucci; I. Ascone; L. Bubacco; M. Beltramini; Di P. Muro; B. Salvato (120-128).
The structural characteristics of oxy- and deoxy-hemocyanins have been investigated using X-ray absorption spectroscopy both in the near-edge (XANES) and for the first shell contribution in the EXAFS region. Several arthropodan and molluscan hemocyanins have been studied in order to trace the inter- and intra-phyla differences. The XANES spectra of oxy-hemocyanins of the different species are remarkably similar, consistent with a very strongly conserved co-ordination geometry of the copper active site. In contrast, small but significant differences are observed between the deoxy-forms of arthropodan and molluscan proteins. In particular, the XANES spectra of deoxy-arthropodan hemocyanins (with the exception of L. polyphemus Hc) show a more intense edge feature at approximately 8983 eV. This difference is tentatively assigned to a more planar geometry of the copper-ligands system in the arthropodan rather than in the molluscan proteins.The first shell analysis of the EXAFS modulation is consistent with the presence of n=3Nε 2 imidazole nitrogens at an average distance of 1.92±0.03 Å from copper in all the deoxy-hemocyanins investigated.Binding of dioxygen results for all hemocyanins in the increase of the number of first shell back-scattering atoms to n=5 with average distances of 1.93 Å. Alternatively, by separating the contribution of Nε 2 imidazole nitrogens and of peroxide O-atoms, n=3 ligands at 1.98±0.03 Å and n=2 ligands at 1.87±0.03 Å are found.
Keywords: Hemocyanins X-ray absorption spectroscopy Dinuclear copper site

The aminopeptidase from Aeromonas proteolytica can function as an esterase by David L. Bienvenue; Rebecca S. Mathew; Dagmar Ringe; Richard C. Holz (129-135).
The aminopeptidase from Aeromonas proteolytica (AAP) can catalyze the hydrolysis of L-leucine ethyl ester (L-Leu-OEt) with a rate of 96±5 s–1 and a K m of 700 µM. The observed turnover number for L-Leu-OEt hydrolysis by AAP is similar to that observed for peptide hydrolysis, which is 67±5 s–1. The k cat values for the hydrolysis of L-Leu-OEt and L-leucine-p-nitroanilide (L-pNA) catalyzed by AAP were determined at different pH values under saturating substrate concentrations. Construction of an Arrhenius plot from the temperature dependence of AAP-catalyzed ester hydrolysis indicates that the rate-limiting step does not change as a function of temperature and is product formation. The activation energy (E a) for the activated ES ester complex is 13.7 kJ mol–1, while the enthalpy and entropy of activation at 25 °C calculated over the temperature range 298–338 K are 11.2 kJ mol–1 and –175 J K–1 mol–1, respectively. The free energy of activation at 25 °C was found to be 63.4 kJ mol–1. The enthalpy of ionization was also measured and was found to be very similar for both peptide and ester substrates, yielding values of 20 kJ mol–1 for L-Leu-OEt and 25 kJ mol–1 for L-pNA. For peptide and L-amino acid ester cleavage reactions catalyzed by AAP, $${{k_{cat}^{H_2 O} } mathord{left/ {vphantom {{k_{cat}^{H_2 O} } {k_{cat}^{D_2 O} }}} ight. kern- ulldelimiterspace} {k_{cat}^{D_2 O} }} = 2.75$$ and 6.07, respectively. Proton inventory data suggest that two protons are transferred in the rate-limiting step of ester hydrolysis while only one is transferred in peptide hydrolysis. The combination of these data with the available X-ray crystallographic, kinetic, spectroscopic, and thermodynamic data for AAP provides new insight into the catalytic mechanism of AAP.
Keywords: Aminopeptidase Aeromonas proteolytica Zinc Kinetics Thermodynamics

Oxalate oxidase catalyzes the oxidation of oxalate to carbon dioxide and hydrogen peroxide, making it useful for clinical analysis of oxalate in biological fluids. An artificial gene for barley oxalate oxidase has been used to produce functional recombinant enzyme in a Pichia pastoris heterologous expression system, yielding 250 mg of purified oxalate oxidase from 5 L of fermentation medium. The recombinant oxalate oxidase was expressed as a soluble, hexameric 140 kDa glycoprotein containing 0.2 g-atom Mn/monomer with a specific activity of 10 U/mg, similar to the properties reported for enzyme isolated from barley. No superoxide dismutase activity was detected in the recombinant oxalate oxidase. EPR spectra indicate that the majority of the manganese in the protein is present as Mn(II), and are consistent with the six-coordinate metal center reported in the recent X-ray crystal structure for barley oxalate oxidase. The EPR spectra change when bulky anions such as iodide bind, indicating conversion to a five-coordinate complex. Addition of oxalate perturbs the EPR spectrum of the Mn(II) sites, providing the first characterization of the substrate complex. The optical absorption spectrum of the concentrated protein contains features associated with a minor six-coordinate Mn(III) species, which disappears on addition of oxalate. EPR spin-trapping experiments indicate that carboxylate free radicals (CO2 ) are transiently produced by the enzyme in the presence of oxalate, most likely during reduction of the Mn(III) sites. These features are incorporated into a turnover mechanism for oxalate oxidase.
Keywords: Pichia pastoris Manganese Germin Oxalate oxidase

Kinetics and active site dynamics of Staphylococcus aureus arsenate reductase by Joris Messens; José C. Martins; Elke Brosens; Karolien Van Belle; Doris M. Jacobs; Rudolph Willem; Lode Wyns (146-156).
Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular arsenate(V) to the more toxic arsenite(III), which is subsequently extruded from the cell. It couples to thioredoxin, thioredoxin reductase and NADPH to be enzymatically active. ArsC is extremely sensitive to oxidative inactivation, has a very dynamic character hampering resonance assignments in NMR and produces peculiar biphasic Michaelis-Menten curves with two V max plateaus. In this study, methods to control ArsC oxidation during purification have been optimized. Next, application of Selwyn's test of enzyme inactivation was applied to progress curves and reveals that the addition of tetrahedral oxyanions (50 mM sulfate, phosphate or perchlorate) allows the control of ArsC stability and essentially eliminates the biphasic character of the Michaelis-Menten curves. Finally, 1H-15N HSQC NMR spectroscopy was used to establish that these oxyanions, including the arsenate substrate, exert their stabilizing effect on ArsC through binding with residues located within a C-X5-R sequence motif, characteristic for phosphotyrosine phosphatases. In view of this need for a tetrahedral oxyanion to structure its substrate binding site in its active conformation, a reappraisal of basic kinetic parameters of ArsC was necessary. Under these new conditions and in contrast to previous observations, ArsC has a high substrate specificity, as only arsenate could be reduced (K m=68 µM, k cat/K m=5.2×104 M–1 s–1), while its product, arsenite, was identified as a mixed inhibitor (K*iu=534 µM, K*ic=377 µM).
Keywords: Arsenate reductase Kinetics NMR spectroscopy Redox enzyme Selwyn test

X-ray absorption spectroscopy (XAS) is used to study ferrous complexes of a bleomycin (BLM) congener, peplomycin (PEP), and two of its derivatives, iso-peplomycin (ISO) and depyruvamide peplomycin (DP), in which potential axial ligands have been perturbed and removed, respectively. Application of extended X-ray absorption fine structure analysis shows an elongation of the short-distance component of the first coordination sphere in DP and ISO relative to PEP. The XAS pre-edge intensity concomitantly decreases with increased axial perturbation. The short-distance component of PEP is correlated to the Fe-pyrimidine bond and is related to the amount of π-back-bonding. Thus, the XAS analysis of these complexes provides structural information relevant to their differences in O2 reactivity.
Keywords: Bleomycin Peplomycin Back-bonding XAS

Nitrite binding to metmyoglobin and methemoglobin in comparison to nitric oxide binding by Alicja Wanat; Joanna Gdula-Argasińska; Dorota Rutkowska-Żbik; Małgorzata Witko; Grażyna Stochel; Rudi van Eldik (165-176).
Nitrite binds reversibly to the ferriheme proteins metmyoglobin and methemoglobin in aqueous buffer solution at a physiological pH of 7.4. The spectral changes recorded for the formation of metMb(NO2 ) differ significantly from those observed for the nitrosylation of metMb, which can be accounted for in terms of the different reaction products. Nitric oxide binding to metMb produces a nitrosyl product with Fe(II)-NO+ character, whereas the reaction with nitrite produces an Fe(III)-NO2 complex. The kinetics of the binding and release of nitrite by metMb and metHb were investigated by stopped-flow techniques at ambient and high pressure. The kinetic traces recorded for the reaction of nitrite with metMb exhibit excellent single-exponential fits, whereas nitrite binding to metHb is characterized by double-exponential kinetics which were assigned to the reactions of the α- and β-chains of metHb with NO2 . The rate constants for the binding of nitrite to metMb and metHb were found to be much smaller than those reported for the binding of NO, such that nitrite impurities will not affect the latter reaction. The activation parameters (ΔH, ΔS ne , ΔV) obtained from the temperature and pressure dependence of the reactions support the operation of a dissociative mechanism for the binding and release of nitrite, similar to that found for the binding and release of NO in metMb.
Keywords: Metmyoglobin Methemoglobin Nitrite Kinetics Activation parameters

Hydrogenases in the "active" state: determination of g-matrix axes and electron spin distribution at the active site by 1H ENDOR spectroscopy by Arnd Müller; Irene Tscherny; Reinhard Kappl; Claude E. Hatchikian; Jürgen Hüttermann; Richard Cammack (177-194).
Hydrons and electrons are substrates for the enzyme hydrogenase, but cannot be observed in X-ray crystal structures. High-resolution 1H electron nuclear double resonance (ENDOR) spectroscopy offers a means to detect the distribution of protons and unpaired electrons. ENDOR spectra were recorded from frozen solutions of the nickel-iron hydrogenases of Desulfovibrio gigas and Desulfomicrobium baculatum, in the "active" state ("Ni-C" EPR signal) and analyzed by orientationally selective simulation methods. The experimental spectra were fitted using a structural model of the nickel-iron centre based on crystallographic results, allowing for differences in electron spin distribution as well as the spatial orientation of the g-matrix (g-tensor), and anisotropic and isotropic hyperfine couplings of the protons nearest to the nickel ion. ENDOR signals, detected after complete deuterium exchange, were assigned to six protons of the cysteines bound to nickel. The assignment took advantage of the substitution of a selenium for a sulfur ligand, which occurs naturally between the [NiFeSe] and [NiFe] hydrogenases from Dm. baculatum and D. gigas, respectively, and was found to affect just two signals. The four signals with the largest hyperfine couplings, including isotropic contributions from 4.5 to 13.5 MHz, were assigned to the β-methylene protons of the two terminal cysteine ligands, one of which is substituted by seleno-cysteine in [NiFeSe] hydrogenase. The electron spin is delocalized onto the nickel (50%) and its sulfur ligands, with a higher proportion on the terminal than the bridging ligands. The g-matrix was found to align with the active site in such a way that the g 1-g 2 plane is nearly coplanar (18.3°) with the plane defined by nickel and three sulfur atoms, and the g 2 axis deviates by 22.9° from the vector between nickel and iron. Significantly for the reaction of the enzyme, direct evidence for the binding of hydrons at the active site was obtained by the detection of H/D-exchangeable ENDOR signals.
Keywords: Hydrogenases Electron nuclear double resonance spectroscopy Electron paramagnetic resonance spectroscopy Nickel Dinuclear centre

Nickel-guanine interactions in DNA: crystal structure of nickel-d[CGTGTACACG]2 by Nicola G. Abrescia; Tam Huynh-Dinh; Juan A. Subirana (195-199).
The aim of this study was to clarify whether Ni2+ ions could bind to guanine bases in a standard B-DNA duplex and eventually induce a B→Z transition. We have determined by X-ray crystallography at 3.1 Å resolution the structure of the alternating deoxynucleotide d(CGTGTACACG), which contains both internal and terminal guanines. The duplex is in the B form. It is shown that nickel ions bind selectively to the N7 atom of guanine 10, which is in an extra-helical position, and guanine 2, which is in the terminal position of the duplex. It does not bind to guanine 4, which lies within a standard B-DNA tract. This simple but unambiguous result proves that nickel ions select between different guanines via steric accessibility. Guanine-Ni2+-guanine bridges among symmetry-related duplexes have also been found. These bridges may explain why Ni2+ ions may act either as a precipitant or a renaturing agent for DNA under certain conditions. The biochemical interaction of nickel with DNA can thus be related to its capacity to specifically bind to B-DNA regions with exposed guanines. Also, from the structural point of view, we have found a terminal cytosine, which forms a C·G:C reverse-Hoogsteen triple structure with a base pair of a neighbor duplex. This type of triplet is seldom found and is here described for the first time for a DNA structure.
Keywords: Oligonucleotide Nickel ion X-ray crystallography DNA

Studies of the reduction and protonation behavior of tetraheme cytochromes using atomic detail by Vitor H. Teixeira; Cláudio M. Soares; António M. Baptista (200-216).
A comparative study of tetraheme cytochrome c 3 molecules from several species was carried out using recently developed theoretical methods based on continuum electrostatics. The binding joint equilibrium of electrons and protons was simulated, revealing the complete thermodynamic aspects of electron-proton coupling in these molecules. The method yields excellent accuracy in terms of midpoint potentials, giving the correct reduction orders in all molecules examined, except for one heme site. The coupling between electrons and protons is shown to be present and significant at physiological pH in all cases. This phenomenon, known as the redox-Bohr effect, though of thermodynamic nature, is shown to have an intrinsic "dynamic" character at the molecular level (in the sense of the empty/occupied fluctuations at the microscopic level), with the binding states of redox and protonatable sites displaying both correlated averages and correlated fluctuations. The protonatable sites more directly involved in the redox-Bohr effect are identified using, among other properties, the statistical correlation between pairs of sites, which automatically reflects indirect effects mediated by other sites. Several sites are identified in this analysis. Propionate D of heme I seems to be the most interesting, generally showing a high correlation not only with its own heme, but also with heme II, corresponding to an indirect stabilization of the reduced forms of both hemes. Other interesting sites are the free histidines of two of the cytochromes and propionate D of heme IV, the latter being potentially associated with redox-induced structural changes. Among the set of cytochromes c 3 analyzed in this study, significant differences are observed for several properties of the acidic cytochrome included in the set, from Desulfovibrio africanus, supporting the hypothesis of a different functional role.
Keywords: Electron-proton coupling Redox-Bohr effect Sulfate-reducing bacteria Continuum electrostatics Binding equilibrium

Observation of an isotope-sensitive low-frequency Raman band specific to metmyoglobin by Shun Hirota; Yasutaka Mizoguchi; Osamu Yamauchi; Teizo Kitagawa (217-221).
A resonance Raman band involving significantly the iron(III)-histidine stretching (νFe-His) character is identified for metmyoglobin (metMb) through isotope sensitivity of its low-frequency resonance Raman bands, but the identification was not successful for methemoglobin (metHb) and its isolated α and β subunits. A band at 218 cm–1 of natural abundance metMb exhibited a low-frequency shift for 15N-His-labeled metMb (–1.4 cm–1 shift), while the strong porphyrin bands at 248 and 271 cm–1 did not shift significantly. The frequency of the 218-cm–1 band of metMb decreased by 1.6 cm–1 in D2O, probably due to Nδ-deuteration of the proximal His, in a similar manner to that of the νFe-His band of deoxyMb in D2O. This 218-cm–1 band shifted slightly to a lower frequency in H2 18O, whereas it did little upon 54Fe isotopic substitution (<0.3 cm–1), presumably because of the six-coordinate structure. The lack of the 54Fe-isotope shift shows that the 218-cm–1 band is specific to metMb and not due to the deoxy species. The intensity of this band decreased for hydroxymetMb and was indiscernible for cyanometMb. For metHb and its α and β subunits, however, the frequencies of the band around 220 cm–1 were not D2O sensitive. These results suggest an assignment of the band around 220 cm–1 to a pyrrole tilting mode, which significantly contains the Fe-His stretching character for metMb but scarcely for metHb and its subunits. The differences in the isotope sensitivity of this band in different proteins are considered to reflect the heme distortion from the planarity and the Fe-His geometry specific to individual proteins.
Keywords: Resonance Raman Iron-histidine stretching character Isotope sensitivity Metmyoglobin Heme protein

Announcement (222-222).

Announcement (223-224).