Ferric Form Research Articles

Probing the Chemotaxis Periplasmic Sensor Domains from Geobacter sulfurreducens by Combined Resonance Raman and Molecular Dynamic Approaches: NO and CO Sensing

The periplasmic sensor domains encoded by genes gsu0582 and gsu0935 are part of methyl accepting chemotaxis proteins in the bacterium Geobacter sulfurreducens (Gs). The sensor domains of these proteins contain a heme-c prosthetic group and a PAS-like fold as revealed by their crystal structures. Biophysical studies of the two domains showed that nitric oxide (NO) binds to the heme in both the ferric and ferrous forms, whereas carbon monoxide (CO) binds only to the reduced form. In order to address these exogenous molecules as possible physiological ligands, binding studies and resonance Raman (RR) spectroscopic characterization of the respective CO and NO adducts were performed in this work. In the absence of exogenous ligands, typical RR frequencies of five-coordinated (5c) high-spin and six-coordinated (6c) low-spin species were observed in the oxidized form. In the reduced state, only frequencies corresponding to the latter were detected. In both sensors, CO binding yields 6c low-spin adducts by replacing the endogenous distal ligand. The binding of NO by the two proteins causes partial disruption of the proximal Fe-His bond, as revealed by the RR fingerprint features of 5cFe-NO and 6cNO-Fe-His species. The measured CO and NO dissociation constants of ferrous GSU0582 and GSU0935 sensors reveal that both proteins have high and similar affinity toward these molecules (K(d) approximately = 0.04-0.08 microM). On the contrary, in the ferric form, sensor GSU0582 showed a much higher affinity for NO (K(d) approximately = 0.3 microM for GSU0582 versus 17 microM for GSU0935). Molecular dynamics calculations revealed a more open heme pocket in GSU0935, which could account for the different affinities for NO. Taken together, spectroscopic data and MD calculations revealed subtle differences in the binding properties and structural features of formed CO and NO adducts, but also indicated a possibility that a (5c) high-spin/(6c) low-spin redox-linked equilibrium could drive the physiological sensing of Gs cells.

Novel Conformational Transitions of Human Cytochrome P450 2C8 during Thermal and Acid‐induced Unfolding

AbstractTransitions among various heme coordination/spin states, heme environments and protein conformations of human cytochrome P450 2C8 were investigated under different denaturing conditions by means of electronic absorption and circular dichroism spectroscopies. It is the first report of it's kind. Our results indicated that the thermal and acid‐induced denaturation could convert P450 2C8 to various P420 forms. In the thermal unfolding process, the ferric P420 thermal form emerged with weakened Fe‐S (thiolate) bond. An absorption band at ca. 425 nm of the ferrous P420 2C8 thermal form was observed, suggesting that the axial Cys435 was protonated or displaced by other ligand. Moreover, the new coordination bond was stabilized when the temperature was cooled down. When binding with CO, the ferrous P420 2C8 thermal form had the protonated thiol of Cys435 as the axial ligand. X‐ray structure of P450 2C8 suggested that the specific structure of the β‐bulge where the axial cysteine ligand located might be the reason of the formation of these P420 2C8 thermal forms. In the acid‐induced unfolding studies, we found that at pH 3.0 the heme could be irreversibly released from the heme pocket of ferric and ferrous P450 2C8. Interestingly, the released heme could form a new coordination bond with an unidentified ligand at the surface of partially unfolded protein when binding with CO at reduced state.

Inhibition Mode of Soybean Lipoxygenase‐1 by Quercetin

Quercetin noncompetitively inhibited the peroxidation of linoleic acid catalyzed by soybean lipoxygenase-1 (EC 1.13.11.12, Type 1) with an IC(50) value of 4.8 microM (1.45 microg/ml). This inhibition is considered to proceed in sequential order, by initially reducing the ferric form of the enzyme to an inactive ferrous form and then, by chelating the iron of the active site of the enzyme. In the pseudoperoxidase assay, quercetin was slowly oxidized by hydroperoxides to a rather stable intermediate, 2-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxybenzofuran-3(2H)-one, and this oxidized intermediate still inhibited the enzymatic oxidation, probably as a chelator. Rutin and kaempferol also exhibited lipoxygenase-1 inhibitory activity, but to a much lesser extent than quercetin.

Electrocatalysis at Microelectrodes: Geometrical Considerations

The influence of the shape of individual electrocatalytically active, axisymmetric particles supported on an inactive substrate, on the current associated with heterogeneous redox reactions involving solution-phase species has been examined theoretically. In agreement with previous work, the diffusion-limited currents, ilim, for a disk and supported truncated spheres of the same area including a full sphere are very similar (within ca. 10%). Large enhancements in the predicted values of ilim were found, however, for spheroids of the prolate type, as the aspect ratio was increased. Analyses of arrays of electrocatalytically active disks embedded in a planar inactive support in the presence of a diffusion boundary layer of well-defined thickness, for example, a rotating disk electrode, revealed that values of ilim very close to those expected for fully catalytic surfaces could be achieved for disk coverages on the order of 1%. Similar conclusions were made for corresponding arrays of small disks embedded in microdisks or spherical inactive supports in quiescent media. This effect might explain observations made for iron porphyrins adsorbed on graphite surfaces in aqueous electrolytes, for which the experimentally determined onset for oxygen reduction is found to be at potentials well ahead of the onset of the voltammetric wave associated with the conversion of the inactive (ferric) to the active (ferrous) form of the macrocycle.

Orientation of the axial ligands and magnetic properties of the hemes in the triheme ferricytochrome PpcA from G. sulfurreducens determined by paramagnetic NMR

The geometry of the axial ligands of the hemes in the triheme cytochrome PpcA from Geobacter sulfurreducens was determined in solution for the ferric form using the unambiguous assignment of the NMR signals of the α-substituents of the hemes. The paramagnetic 13C shifts of the hemes can be used to define the heme electronic structure, the geometry of the axial ligands, and the magnetic susceptibility tensor. The latter establishes the magnitude and geometrical dependence of the pseudocontact shifts, which are crucial to warrant reliable structural constraints for a detailed structural characterization of this paramagnetic protein in solution.

The role of iron uptake in pathogenicity and symbiosis in Photorhabdus luminescens TT01

BackgroundPhotorhabdus are Gram negative bacteria that are pathogenic to insect larvae whilst also having a mutualistic interaction with nematodes from the family Heterorhabditis. Iron is an essential nutrient and bacteria have different mechanisms for obtaining both the ferrous (Fe2+) and ferric (Fe3+) forms of this metal from their environments. In this study we were interested in analyzing the role of Fe3+ and Fe2+ iron uptake systems in the ability of Photorhabdus to interact with its invertebrate hosts.ResultsWe constructed targeted deletion mutants of exbD, feoABC and yfeABCD in P. luminescens TT01. The exbD mutant was predicted to be crippled in its ability to obtain Fe3+ and we show that this mutant does not grow well in iron-limited media. We also show that this mutant was avirulent to the insect but was unaffected in its symbiotic interaction with Heterorhabditis. Furthermore we show that a mutation in feoABC (encoding a predicted Fe2+ permease) was unaffected in both virulence and symbiosis whilst the divalent cation transporter encoded by yfeABCD is required for virulence in the Tobacco Hornworm, Manduca sexta (Lepidoptera) but not in the Greater Wax Moth, Galleria mellonella (Lepidoptera). Moreover the Yfe transporter also appears to have a role during colonization of the IJ stage of the nematode.ConclusionIn this study we show that iron uptake (via the TonB complex and the Yfe transporter) is important for the virulence of P. luminescens to insect larvae. Moreover this study also reveals that the Yfe transporter appears to be involved in Mn2+-uptake during growth in the gut lumen of the IJ nematode. Therefore, the Yfe transporter in P. luminescens TT01 is important during colonization of both the insect and nematode and, moreover, the metal ion transported by this pathway is host-dependent.

Structure and Properties of a Bis-Histidyl Ligated Globin from Caenorhabditis elegans,

Globins are heme-containing proteins that are best known for their roles in oxygen (O(2)) transport and storage. However, more diverse roles of globins in biology are being revealed, including gas and redox sensing. In the nematode Caenorhabditis elegans, 33 globin or globin-like genes were recently identified, some of which are known to be expressed in the sensory neurons of the worm and linked to O(2) sensing behavior. Here, we describe GLB-6, a novel globin-like protein expressed in the neurons of C. elegans. Recombinantly expressed full-length GLB-6 contains a heme site with spectral features that are similar to those of other bis-histidyl ligated globins, such as neuroglobin and cytoglobin. In contrast to these globins, however, ligands such as CO, NO, and CN(-) do not bind to the heme in GLB-6, demonstrating that the endogenous histidine ligands are likely very tightly coordinated. Additionally, GLB-6 exhibits rapid two-state autoxidation kinetics in the presence of physiological O(2) levels as well as a low redox potential (-193 +/- 2 mV). A high-resolution (1.40 A) crystal structure of the ferric form of the heme domain of GLB-6 confirms both the putative globin fold and bis-histidyl ligation and also demonstrates key structural features that can be correlated with the unusual ligand binding and redox properties exhibited by the full-length protein. Taken together, the biochemical properties of GLB-6 suggest that this neural protein would most likely serve as a physiological sensor for O(2) in C. elegans via redox signaling and/or electron transfer.

Expression and characterization of Mycobacterium tuberculosis CYP144: Common themes and lessons learned in the M. tuberculosis P450 enzyme family

CYP144 from Mycobacterium tuberculosis was expressed and purified. CYP144 demonstrates heme thiolate coordination in its ferric form, but the cysteinate is protonated to thiol in both the carbon monoxide-bound and ligand-free ferrous forms (forming P420 in the former). Tight binding of various azole drugs was shown, with affinity for miconazole ( K d = 0.98 μM), clotrimazole (0.37 μM) and econazole (0.78 μM) being highest. These azoles are also the trio with the highest affinity for the essential CYP121 and for the cholesterol oxidase CYP125 (essential for host infection), and have high potency as anti-mycobacterial drugs. Construction of a Mtb gene knockout strain demonstrated that CYP144 is not essential for growth in vitro. However the deletion strain was more sensitive to azole inhibition in culture suggesting an important role for CYP144 in cell physiology and/or in mediating azole resistance. The biophysical and genetic features of CYP144 are compared to those of other characterized Mtb P450s, identifying both commonality in properties (including thiolate protonation in ferrous P450s) and intriguing differences in thermodynamic and spectroscopic features. Our developing knowledge of the Mtb P450s has revealed unusual biochemistry and gene essentiality, highlighting their potential as drug targets in this human pathogen.

α-Hemoglobin Stabilizing Protein (AHSP), a Kinetic Scheme of the Action of a Human Mutant, AHSPV56G

A kinetic analysis has been made of the interaction of alpha-Hb chains with a mutant alpha-hemoglobin stabilizing protein, AHSP(V56G), which is the first case of an AHSP mutation associated with clinical symptoms of mild thalassemia syndrome. The chaperone AHSP is thought to protect nascent alpha chains until final binding to the partner beta-Hb. Rather than protecting alpha chains, the mutant chaperone is partially unfolded but recovers its secondary structure via interaction with alpha-Hb. For both AHSP(WT) and AHSP(V56G), the binding to alpha-Hb is quite rapid relative to the alpha-beta reaction, as expected because the chaperone binding must be quite competitive to complete the alpha chain folding process before alpha-Hb binds irreversibly to beta-Hb. The main kinetic difference is a dissociation rate of AHSP(V56G).alpha-Hb some four times faster relative to AHSP.alpha-Hb. Considering a role of protein folding, the AHSP(V56G) apparently does not bind long enough (0.5 s versus 2 s for the WT) to complete the structural modifications. The overall replacement reaction (AHSP.alpha-Hb + beta-Hb --> AHSP + alphabeta) can be quite long, especially if there is an excess of AHSP relative to beta-Hb monomers.

Plasma membrane ferric reductase activity of iron-limited algal cells is inhibited by ferric chelators

Iron-limited cells of the green alga Chlorella kesslerii use a reductive mechanism to acquire Fe(III) from the extracellular environment, in which a plasma membrane ferric reductase reduces Fe(III)-chelates to Fe(II), which is subsequently taken up by the cell. Previous work has demonstrated that synthetic chelators both support ferric reductase activity (when supplied as Fe(III)-chelates) and inhibit ferric reductase. In the present set of experiments we extend these observations to naturally-occurring chelators and their analogues (desferrioxamine B mesylate, schizokinen, two forms of dihydroxybenzoic acid) and also two formulations of the commonly-used herbicide N-(phoshonomethyl)glycine (glyphosate). The ferric forms of the larger siderophores (desferrioxamine B mesylate, schizokinen) and Fe(III)-N-(phoshonomethyl)glycine (as the isopropylamine salt) all supported rapid rates of ferric reductase activity, while the iron-free forms inhibited reductase activity. The smaller siderophores/siderophore precursors, 2,3- and 3,4-dihydroxybenzoic acids, did not support high rates of reductase in the ferric form but did inhibit reductase activity in the iron-free form. Bioassays indicated that Fe(III)-chelates that supported high rates of ferric reductase activity also supported a large stimulation in the growth of iron-limited cells, and that an excess of iron-free chelator decreased the growth rate. With respect to N-(phosphonomethyl)glycine, there were differences between the pure compound (free acid form) and the most common commercial formulation (which also contains isopropylamine) in terms of supporting and inhibiting ferric reductase activity and growth. Overall, these results suggest that photosynthetic organisms that use a reductive strategy for iron acquisition both require, and are potentially simultaneously inhibited by, ferric chelators. Furthermore, these results also may provide an explanation for the frequently contradictory results of N-(phosphonomethyl)glycine application to crops: we suggest that low concentrations of this molecule likely solubilize Fe(III), making it available for plant growth, but that higher (but sub-lethal) concentrations decrease iron acquisition by inhibiting ferric reductase activity.

The heme pocket of the globin domain of the globin-coupled sensor of Geobacter sulfurreducens — An EPR study

The globin-coupled sensor (GCS) of Geobacter sulfurreducens is unique amongst GCSs in that its signalling domain is a transmembrane domain with yet unknown function. In the present work we use X-band continuous-wave and pulsed electron paramagnetic resonance (EPR) to investigate the ferric form of the globin domain of the G. sulfurreducens GCS ( GsGCS 162) at pH 8.5. This form shows a unique bis-histidine coordination of the heme with the F8His and E11His. In contrast with previous crystal structure data, where three conformers of the heme structure were identified, ferric GsGCS 162 assumes only one conformation in frozen solution. The EPR data of ferric GsGCS162 are compared in detail with those of other bis-histidine coordinated globins, including other GCS systems.

Characterization of conformational changes and noncovalent complexes of myoglobin by electrospray ionization mass spectrometry, circular dichroism and fluorescence spectroscopy

Electrospray ionization mass spectrometry (ESI-MS) was employed to monitor the heme release and the conformational changes of myoglobin (Mb) under different solvent conditions, and to observe ligand bindings of Mb. ESI-MS, complemented by circular dichroism and fluorescence spectroscopy, was used to study the mechanism of acid- and organic solvent-induced denaturation by probing the changes in the secondary and the tertiary structure of Mb. The results obtained show that complete disruption of the heme-protein interactions occurs when Mb is subjected to one of the following solution conditions: pH 3.2-3.6, or solution containing 20-30% acetonitrile or 40-50% methanol. Outside these ranges, Mb is present entirely in its native state (binding with a heme group) or as apomyoglobin (i.e. without the heme). Spectroscopic data demonstrate that the denaturation mechanism of Mb induced by acid may be significantly different from that by the organic solvent. Low pH reduces helices in Mb, whereas certain organic content level in solution results in the loss of the tertiary structure. ESI-MS conditions were established to observe the H(2)O- and CO-bound Mb complexes, respectively. H(2)O binding to metmyoglobin (17,585 Da), where the heme iron is in the ferric oxidation state, is observed in ESI-MS. CO binding to Mb (17,595 Da), on the other hand, can be only observed after the heme iron is reduced to the ferrous form. Therefore, ESI-MS combined with spectroscopic techniques provides a useful means for probing the formation of ligand-binding complexes and characterizing protein conformational changes.

A density functional investigation of hydrogen peroxide activation by high-valent heme centers: implications for the catalase catalytic cycle

Catalases employ a tyrosinate-ligated ferric heme in order to catalyze the dismutation of hydrogen peroxide to O2 and water. In the first half of the catalytic cycle, H2O2 oxidizes Fe(III) to the formally Fe(V) state commonly referred to as Compound I. The second half of the cycle entails oxidation of a second hydrogen peroxide molecule by Compound I to dioxygen. The present study employs density functional (DFT) calculations to examine the nature of this second step of the catalatic reaction. In order to account for the unusual choice of tyrosinate as an axial ligand in catalases, oxidation of hydrogen peroxide by an imidazole-ligated Compound I is also examined, bearing in mind that imidazole-ligated hemoproteins such as myoglobin or horseradish peroxidase tend to display little, if any, catalatic activity. Furthermore, in order to gauge the importance of the cation radical of Compound I in peroxide activation, the performance of Compound II (the one-electron reduced version of Compound I, formally Fe(IV) ), is also examined. It is found that hydrogen peroxide oxidation occurs in a quasi-concerted manner, with two hydrogen-atom transfer reactions, and that the tyrosinate ligand is in no way required at this stage. We propose that the role of the tyrosinate is purely thermodynamic, in avoiding accumulation of the much less peroxide-reactive ferrous form in vivo – all in line with the predominantly thermodynamic role of the cysteinate ligands in enzymes such as cytochromes P450.

ROLE OF CHELATABLE IRON VERSUS MYOGLOBIN IN OXIDATIVE STRESS AFTER CRUSH TRAUMA

ROLE OF CHELATABLE IRON VERSUS MYOGLOBIN IN OXIDATIVE STRESS AFTER CRUSH TRAUMA

Ascorbate enhances iron uptake into intestinal cells through formation of a FeCl 3–ascorbate complex

It has been well documented that ascorbate enhances iron uptake, with a proposed mechanism based on reduction to the more absorbable ferrous form. We have performed a study on the effects of ascorbate on ferric iron uptake in the human epithelial Caco-2 cell-line. Ascorbate increased uptake in a concentration-dependent manner with a significant difference between iron uptake and reduction. Uptake kinetics are characteristic of a non-essential activator and the formation of an Fe 3+–ascorbate complex. This investigation provides evidence that ascorbate enhances the apical uptake of ferric iron into Caco-2 cells through the formation of a Fe 3+–ascorbate complex.

Abstract 1470: 15-Deoxy-Δ12,14-prostaglandin J2 up-regulates expression of p53 and p21 through Nrf2-driven heme oxygenase-1 induction in human breast cancer (MCF-7) cells

Abstract Heme oxygenase-1 (HO-1) is a stress-responsive enzyme that has antioxidant and cytoprotective functions. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), an endogenous ligand of peroxisome proliferator-activated receptor γ (PPARγ), has been reported to induce HO-1 and other antioxidant enzymes in certain cell lines. In the present work, we investigated the effects of HO-1 on the expression of p53 and p21 induced by 15d-PGJ2 in human breast cancer (MCF-7) cells. Treatment of MCF-7 cells with 15d-PGJ2 (30 μM) led to time-dependent increases in the expression of p53 and p21 as well as HO-1. The up-regulation of p53 and p21 expression by 15d-PGJ2 was abrogated by pharmacological inhibition with zinc protophophyrin (10 μM) or si-RNA knock down of HO-1. Nrf2, a basic-leucine zipper transcription factor, plays a key role in regulating the up-regulation of many antioxidant enzymes including HO-1. In MCF-7 cells transfected with NRF2 si-RNA, 15d-PGJ2 failed to induce expression of p53, p21 and HO-1. In addition, HO-1 inducers, such as cobalt protoporphyrin (25 μM) and hemin (10 μM), upregulated the expression of p53 and p21 without eliciting any apparent cytotoxicity. HO-1 degrades heme to produce iron, biliverdin, and carbon monoxide. We hypothesize that iron, a by-product of HO-1-catalyzed reactions, could mediate 15d-PGJ2-induced expression p53 and p21. Up-regulation of p53 and p21 expression by 15d-PGJ2 was abrogated by the iron chelator desferrioxamine (25 μM) in MCF-7 cells. Iron released from heme by HO-1 activity is mostly in the ferrous (Fe2+) form. When MCF-7 cells were treated with the iron-specific chelator phenanathroline (10 μM), 15d-PGJ2-induced expression of p53 and p21 was attenuated. However, p53 induction by 15d-PGJ2 was not affected by GW9662, a PPARγ antagonist. In addition, levels of the iron-sequestering protein H-ferritin were induced by 15d-PGJ2 in MCF-7 cells. Addition of the antioxidant N-acetylcysteine lowered the 15d-PGJ2-mediated H-ferritin levels. Recently, it has been reported that p21 as a positive regulator of Nrf2 extends the prosurvival function of p53 through recruitment of stress-response genes. Up-regulation of p53 and p21 expression via Nrf2-driven HO-1 induction and subsequent release of iron together with elevated expression of H-ferritin may confer resistance to oxidative damage in MCF-7 cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1470.

Globin-like proteins in Caenorhabditis elegans: in vivo localization, ligand binding and structural properties

BackgroundThe genome of the nematode Caenorhabditis elegans contains more than 30 putative globin genes that all are transcribed. Although their translated amino acid sequences fit the globin fold, a variety of amino-acid substitutions and extensions generate a wide structural diversity among the putative globins. No information is available on the physicochemical properties and the in vivo expression.ResultsWe expressed the globins in a bacterial system, characterized the purified proteins by optical and resonance Raman spectroscopy, measured the kinetics and equilibria of O2 binding and determined the crystal structure of GLB-1* (CysGH2 → Ser mutant). Furthermore, we studied the expression patterns of glb-1 (ZK637.13) and glb-26 (T22C1.2) in the worms using green fluorescent protein technology and measured alterations of their transcript abundances under hypoxic conditions.GLB-1* displays the classical three-over-three α-helical sandwich of vertebrate globins, assembled in a homodimer associated through facing E- and F-helices. Within the heme pocket the dioxygen molecule is stabilized by a hydrogen bonded network including TyrB10 and GlnE7.GLB-1 exhibits high ligand affinity, which is, however, lower than in other globins with the same distal TyrB10-GlnE7 amino-acid pair. In the absence of external ligands, the heme ferrous iron of GLB-26 is strongly hexacoordinated with HisE7, which could explain its extremely low affinity for CO. This globin oxidizes instantly to the ferric form in the presence of oxygen and is therefore incapable of reversible oxygen binding.ConclusionThe presented data indicate that GLB-1 and GLB-26 belong to two functionally-different globin classes.

Index of Suspicion

Index of Suspicion

Arsenopyrite dissolution rates in O2-bearing solutions

Abstract Arsenopyrite dissolution was studied by means of long-term, stirred and non-stirred flow-through experiments in the pH range of 1 to 9 at 25, 50 and 70 °C and at different input dissolved-O2 concentrations (from 0.2 to 8.7 mg L− 1). At pH lower than 4, aqueous iron, which is mainly in the ferrous form, and arsenic are stoichiometrically released. Sulphur concentrations released were lower than stoichiometrically expected (S/As In the light of these results, the steady-state dissolution rates were estimated by the release of arsenic at pH R arsenopyrite mol m − 2 s − 1 25 ° C = 10 − 7.41 ± 0.47 ⋅ a O 2 0.76 ± 0.11 ⋅ a H + − 0.12 ± 0.07 where aO2 and aH+ are the activities of hydrogen ions and dissolved oxygen, respectively and their exponents were estimated from multiple linear regression of the dissolution rates. Temperature increase from 25 to 70 °C yields an apparent activation energy for the arsenopyrite oxidation by dissolved oxygen of 18.5 ± 1.6 kJ mol− 1. At pH > 6, aqueous iron is mainly in the ferric form and is depleted as it precipitates as Fe-oxyhydroxide onto arsenopyrite surfaces, yielding Fe/As and Fe/S less than one; between pHs 7 and 9, iron depletion is complete, and sulphur released is more abundant than arsenic released, which is precipitated as As–O phases, as confirmed by MicroRaman spectroscopy. At pHs 6–9, iron-oxyhydroxide phases and arsenic oxide phases upon the arsenopyrite surface provide an effective layer that reduces diffusion of dissolved oxygen and arsenopyrite dissolution. As coating on the arsenopyrite surface becomes the rate-limiting step, the Shrinking Core Model (SCM) allows quantification of the surface dissolution rate, especially from data obtained where the effect of coating was still negligible. The SCM also allowed us to calculate the effective coefficient for oxygen diffusion through the coating, which can vary from 10− 17 to 1.5 · 10− 16 m2 s− 1. The formation of such a coating produced a decrease in arsenic and sulphur release over time and a final surface passivation.

Biochemical and cellular investigation of Vitreoscilla hemoglobin (VHb) variants possessing efficient peroxidase activity.

Peroxidase-like activity of Vitreoscilla hemoglobin (VHb) has been recently disclosed. To maximize such activity, two catalytically conserved residues (histidine and arginine) found in distal pocket of peroxidases have successfully been introduced into that of the VHb. Fifteen-fold increase in catalytic constant (k(cat)) was obtained in P54R variant, which was presumably attributable to the lower rigidity and higher hydrophilicity of distal cavity arising from substitution of proline to arginine. None of the modifications altered the affinity towards either H(2)O(2) or ABTS substrate. Spectroscopic studies revealed that VHb variants harboring T29H mutation apparently demonstrated the spectral shift in both ferric and ferrous forms (406-408 to 411 nm and 432 to 424-425 nm, respectively). All VHb proteins in ferrous state had lambda(soret) peak at approximately 419 nm following the carbon monoxide (CO) binding. Expression of P54R mutant mediated the down-regulation of iron superoxide dismutase (FeSOD) as identified by 2-dimensional gel electrophoresis (2-DE) and peptide mass fingerprinting (PMF). According to the high peroxidase activity of P54R, it could effectively eliminate autoxidation-derived H2O2, which is a cause of heme degradation and iron release. This decreased the iron availability and consequently reduced the formation of Fe(2+)-ferric uptake regulator protein (Fe(2+)-Fur), an inducer of FeSOD expression.