Mössbauer identification of a protonated ferryl species in catalase from Proteus mirabilis: density functional calculations on related models

The Proteus mirabilis catalase is one of the most efficient heme-containing catalase and forms a relatively stable compound II. Samples of compound II were prepared from PMC enriched in (57)Fe. For the first time, two different forms of compound II, namely low pH compound II (LpH II) (43%) and high pH compound II (HpH II) (25%), have been characterized by M?ssbauer spectroscopy at pH 8.3. The ratio LpH II/HpH II increases irreversibly with decreasing pH. The large quadrupole splitting value of LpH II (DeltaE(Q)=2.29 (2) mm/s, with delta(/Fe)=0.03 (2) mm/s), compared to that of HpH II (DeltaE(Q)=1.47 (2) mm/s, with delta(/Fe)=0.07 (2) mm/s), reflects the protonation of the ferryl group. Quadrupole splitting values of 1.46 and 2.15mm/s have been computed by DFT for optimized models of the ferryl compound II (model 1) and the protonated ferryl compound II (model 2), respectively, starting from the Fe(IV)O model initially published by Rovira and Fita [C. Rovira, I. Fita, J. Phys. Chem. B 107 (2003) 5300-5305]. Therefore, we attribute the LpH II compound to a protonated ferryl Fe(IV)-OH complex, whereas the HpH II compound corresponds to the classical ferryl Fe(IV)O complex.     

On the status of ferryl protonation

We examine the issue of ferryl protonation in heme proteins. An analysis of the results obtained from X-ray crystallography, resonance Raman spectroscopy, and extended X-ray absorption spectroscopy (EXAFS) is presented. Fe-O bond distances obtained from all three techniques are compared using Badger's rule. The long Fe-O bond lengths found in the ferryl crystal structures of myoglobin, cytochrome c peroxidase, horseradish peroxidase, and catalase deviate substantially from the values predict by Badger's rule, while the oxo-like distances obtained from EXAFS measurements are in good agreement with the empirical formula. Density functional calculations, which suggest that M?ssbauer spectroscopy can be used to determine ferryl protonation states, are presented. Our calculations indicate that the quadrupole splitting (DeltaE(Q)) changes significantly upon ferryl protonation. New resonance Raman data for horse-heart myoglobin compound II (Mb-II, pH 4.5) are also presented. An Fe-O stretching frequency of 790cm(-1) (shifting to 754cm(-1) with (18)O substitution) was obtained. This frequency provides a Badger distance of r(Fe-O)=1.66A. This distance is in agreement with the 1.69A Fe-O bond distance obtained from EXAFS measurements but is significantly shorter than the 1.93A bond found in the crystal structure of Mb-II (pH 5.2). In light of the available evidence, we conclude that the ferryl forms of myoglobin (pKa4), horseradish peroxidase (pKa4), cytochrome c peroxidase (pKa4), and catalase (pKa7) are not basic. They are authentic Fe(IV)oxos with Fe-O bonds on the order of 1.65A.     

Hydrogen-bonding conformations of tyrosine B10 tailor the hemeprotein reactivity of ferryl species

Ferryl compounds [Fe(IV)=O] in living organisms play an essential role in the radical catalytic cycle and degradation processes of hemeproteins. We studied the reactions between H₂O₂ and hemoglobin II (HbII) (GlnE7, TyrB10, PheCD1, PheE11), recombinant hemoglobin I (HbI) (GlnE7, PheB10, PheCD1, PheE11), and the HbI PheB10Tyr mutant of L. pectinata. We found that the tyrosine residue in the B10 position tailors, in two very distinct ways, the reactivity of the ferryl species, compounds I and II. First, increasing the reaction pH from 4.86 to 7.50, and then to 11.2, caused the the second-order rate constant for HbII to decrease from 141.60 to 77.78 M⁻¹ s⁻¹, and to 2.96 M⁻¹ s⁻¹, respectively. This pH dependence is associated with the disruption of the heme–tyrosine (603 nm) protein moiety, which controls the access of the H₂O₂ to the hemeprotein active center, thus regulating the formation of the ferryl species. Second, the presence of compound I was evident in the UV–vis spectra (648-nm band) in the reactions of HbI and recombinant HbI with H₂O₂, This band, however, is completely absent in the analogous reaction with HbII and the HbI PheB10Tyr mutant. Therefore, the existence of a hydrogen-bonding network between the heme pocket amino acids (i.e., TyrB10) and the ferryl compound I created a path much faster than 3.0×10⁻² s⁻¹ for the decay of compound I to compound II. Furthermore, the decay of the heme ferryl compound I to compound II was independent of the proximal HisF8 trans-ligand strength. Thus, the pH dependence of the heme–tyrosine moiety complex determined the overall reaction rate of the oxidative reaction limiting the interaction with H₂O₂ at neutral pH. The hydrogen-bonding strength between the TyrB10 and the heme ferryl species suggests the presence of a cycle where the ferryl consumption by the ferric heme increases significantly the pseudoperoxidase activity of these hemeproteins.     

Characterization of a modified nitrogenase Fe protein from Klebsiella pneumoniae in which the 4Fe4S cluster has been replaced by a 4Fe4Se cluster

The Azotobacter vinelandii nifS gene product has been used with selenocysteine to reconstitute Klebsiella pneumoniae nitrogenase Fe protein. Chemical analysis and extended X-ray absorption fine structure (EXAFS) spectroscopy show that the 4Fe4S cluster present in the native protein is replaced by a 4Fe4Se cluster. As well, EXAFS spectroscopy shows that the bond lengths to the cysteine thiolate ligands shrink by 0.05 Å (from 2.28 to 2.23 Å) upon reduction, whereas the Fe–Fe distance is essentially unchanged. Thus, the core of the 4Fe4Se cluster remains essentially static on reduction, whilst the external cysteine thiolate ligands are pulled in towards the cluster. Compared with native (S)–Fe protein, the (Se)–Fe protein has a 20-fold increased rate of MgATP-induced Fe chelation, a sixfold decreased specific activity for acetylene reduction, a fivefold decreased rate of MgATP-dependent electron transfer from (Se)–Fe protein to MoFe protein, and a fourfold increase in the ATP to 2e ^? ratio. The high ATP to 2e ^? ratio and decreased specific activity are consistent with a lower rate of dissociation of oxidized (Se)–Fe protein from reduced MoFe protein. Thus, the relatively small adjustments in the Fe protein structure necessary to accommodate the 4Fe4Se cluster are transmitted both to adjacent residues that dock at the surface of the MoFe protein and to the ATP hydrolysis sites located approximately 19 Å away.     

Binding of VIVO2+ to the Fe binding sites of human serum transferrin. A theoretical study

The binding of V^IVO²⁺ to human serum transferrin (hTF) at the Fe^III binding sites is addressed. Geometry optimization calculations were performed for the binding of V^IVO²⁺ to the N-terminal lobe of hTF (hTF_N), and indicate that in the presence of CO₃ ^2? or HCO₃ ^?, V^IV is bound to five atoms in a distorted geometry. The structures of V^IVO–hTF_N species optimized at the semiempirical level were also used to calculate the ⁵¹V and ¹⁴N A tensors by density functional theory methods, and were compared with the reported experimental values. Globally, of all the calculated V^IVO–hTF structures, the one that yields the lowest calculated heats of formation and minimum deviations from the experimental values of the ⁵¹V and ¹⁴N A tensor components is the structure that includes CO₃ ^2? as a synergistic anion. In this structure the V=O bond length is approximately 1.6 Å, and the vanadium atom is also coordinated to the phenolate oxygen atom of Tyr188 (at approximately 1.9 Å), the aspartate oxygen atom of Asp63 (at approximately 1.9 Å), the His249 Nτ atom (at approximately 2.1 Å), and a carbonate oxygen atom (at approximately 1.8 Å). The Tyr95 phenolic ocygen atom is approximately 3.3 Å from the metal center, and thus is very weakly bound to V^IV. All of these oxygen atoms are able to establish dipolar interactions with groups of the protein.     

Unprecedented access of phenolic substrates to the heme active site of a catalase: Substrate binding and peroxidase‐like reactivity of Bacillus pumilus catalase monitored by X‐ray crystallography and EPR spectroscopy

Heme‐containing catalases and catalase‐peroxidases catalyze the dismutation of hydrogen peroxide as their predominant catalytic activity, but in addition, individual enzymes support low levels of peroxidase and oxidase activities, produce superoxide, and activate isoniazid as an antitubercular drug. The recent report of a heme enzyme with catalase, peroxidase and penicillin oxidase activities in Bacillus pumilus and its categorization as an unusual catalase‐peroxidase led us to investigate the enzyme for comparison with other catalase‐peroxidases, catalases, and peroxidases. Characterization revealed a typical homotetrameric catalase with one pentacoordinated heme b per subunit (Tyr340 being the axial ligand), albeit in two orientations, and a very fast catalatic turnover rate (k_cat = 339,000 s^?1). In addition, the enzyme supported a much slower (k_cat = 20 s^?1) peroxidatic activity utilizing substrates as diverse as ABTS and polyphenols, but no oxidase activity. Two binding sites, one in the main access channel and the other on the protein surface, accommodating pyrogallol, catechol, resorcinol, guaiacol, hydroquinone, and 2‐chlorophenol were identified in crystal structures at 1.65–1.95 Å. A third site, in the heme distal side, accommodating only pyrogallol and catechol, interacting with the heme iron and the catalytic His and Arg residues, was also identified. This site was confirmed in solution by EPR spectroscopy characterization, which also showed that the phenolic oxygen was not directly coordinated to the heme iron (no low‐spin conversion of the Fe^III high‐spin EPR signal upon substrate binding). This is the first demonstration of phenolic substrates directly accessing the heme distal side of a catalase. Proteins 2015; 83:853–866. © 2015 Wiley Periodicals, Inc.     

Verdoheme formation in Proteus mirabilis catalase

Background

Heme oxidative degradation has been extensively investigated in peroxidases but not in catalases. The verdoheme formation, a product of heme oxidation which inactivates the enzyme, was studied in Proteus mirabilis catalase.

Methods

The verdoheme was generated by adding peracetic acid and analyzed by mass spectrometry and spectrophotometry.

Results

Kinetics follow-up of different catalase reactional intermediates shows that i) the formation of compound I always precedes that of verdoheme, ii) compound III is never observed, iii) the rate of compound II decomposition is not compatible with that of verdoheme formation, and iv) dithiothreitol prevents the verdoheme formation but not that of compound II, whereas NADPH prevents both of them. The formation of verdoheme is strongly inhibited by EDTA but not increased by Fe³⁺ or Cu²⁺ salts. The generation of verdoheme is facilitated by the presence of protein radicals as observed in the F194Y mutated catalase. The inability of the inactive variant (H54F) to form verdoheme, indicates that the heme oxidation is fully associated to the enzyme catalysis.

Conclusion

These data, taken together, strongly suggest that the verdoheme formation pathway originates from compound I rather than from compound II.

General significance

The autocatalytic verdoheme formation is likely to occur in vivo.     

Neutrophilic, nitrate-dependent, Fe(II) oxidation by a Dechloromonas species

A species of Dechloromonas, strain UWNR4, was isolated from a nitrate-reducing, enrichment culture obtained from Wisconsin River (USA) sediments. This strain was characterized for anaerobic oxidation of both aqueous and chelated Fe(II) coupled to nitrate reduction at circumneutral pH. Dechloromonas sp. UWNR4 was incubated in anoxic batch reactors in a defined medium containing 4.5–5 mM NO₃ ^?, 6 mM Fe²⁺ and 1–1.8 mM acetate. Strain UWNR4 efficiently oxidized Fe²⁺ with 90 % oxidation of Fe²⁺ after 3 days of incubation. However, oxidation of Fe²⁺ resulted in Fe(III)-hydroxide-encrusted cells and loss of metabolic activity, suggested by inability of the cells to utilize further additions of acetate. In similar experiments with chelated iron (Fe(II)-EDTA), encrusted cells were not produced and further additions of acetate and Fe(II)-EDTA could be oxidized. Although members of the genus Dechloromonas are primarily known as perchlorate and nitrate reducers, our findings suggest that some species could be members of microbial communities influencing iron redox cycling in anoxic, freshwater sediments. Our work using Fe(II)-EDTA also demonstrates that Fe(II) oxidation was microbially catalyzed rather than a result of abiotic oxidation by biogenic NO₂ ^?.     

Acidic pH conditions induce dissociation of the haem from the protein and destabilise the catalase isolated from <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

The stability (half-life, t_½) of the large catalase (CAT) isolated from Aspergillus terreus was decreased under acidic conditions (maximum t_½ ~8.5 months at pH ≤ 6) versus alkaline conditions (t_½ ~15 months at pH 8–12). Acidic conditions induce the dissociation of haem from CAT, as revealed from a reduction in the Soret peak intensity at 405 nm and an increase in the peak current at Fe³⁺/Fe²⁺ redox potentials. This increase in current is attributed to the facile electron transfer from the free haem generated on the electrode surface as a result of its disintegration from the insulating protein matrix. The haem isolated from CAT at acidic condition was reconstituted with apo-CAT at alkaline denaturing conditions to regenerate the CAT activity.     

Warfarin modulates the nitrite reductase activity of ferrous human serum heme–albumin

Human serum heme–albumin (HSA–heme–Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA–heme–Fe [HSA–heme–Fe(II)] is reported. The value of the second-order rate constant for the reduction of $ {\text{NO}}_{2}^{ - } $ to NO and the concomitant formation of nitrosylated HSA–heme–Fe(II) (i.e., k _on) is 1.3 M^?1 s^?1 at pH 7.4 and 20 °C. Values of k _on increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA–heme–Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA–heme–Fe(II) is (3.1 ± 0.4) × 10^?4 M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the $ {\text{NO}}_{2}^{ - } $ reductase activity of HSA–heme–Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(–heme–Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity.     

Superoxide radical inhibits catalase

Catalase was inhibited by a flux of O2- generated in situ by the aerobic xanthine oxidase reaction. Two distinct types of inhibition could be distinguished. One of these was rapidly established and could be as rapidly reversed by the addition of superoxide dismutase. The second developed slowly and was reversed by ethanol, but not by superoxide dismutase. The rapid inhibition was probably due to conversion of catalase to the ferrooxy state (compound III), while the slow inhibition was due to conversion to the ferryl state (compound II). Since neither compound III nor compound II occurs in the catalatic reaction pathway, they are inactive. This inhibition of catalase by O2- provides the basis for a synergism between superoxide dismutase and catalase. Such synergisms have been observed in vitro and may be significant in vivo.     

Quantifying the contribution of land use to N<Subscript>2</Subscript>O,NO and CO<Subscript>2</Subscript> fluxes in a montane forest ecosystem of Kenya

Increasing demand for food and fibre by the growing human population is driving significant land use (LU) change from forest into intensively managed land systems in tropical areas. But empirical evidence on the extent to which such changes affect the soil-atmosphere exchange of trace gases is still scarce, especially in Africa. We investigated the effect of LU on soil trace gas production in the Mau Forest Complex region, Kenya. Intact soil cores were taken from natural forest, commercial and smallholder tea plantations, eucalyptus plantations and grazing lands, and were incubated in the lab under different soil moisture conditions. Soil fluxes of nitrous oxide (N₂O), nitric oxide (NO) and carbon dioxide (CO₂) were quantified, and we approximated annual estimates of soil N₂O and NO fluxes using soil moisture values measured in situ. Forest and eucalyptus plantations yielded annual fluxes of 0.3–1.3 kg N₂O–N ha^?1 a^?1 and 1.5–5.2 kg NO–N ha^?1 a^?1. Soils of commercial tea plantations, which are highly fertilized, showed higher fluxes (0.9 kg N₂O–N ha^?1 a^?1 and 4.3 kg NO–N ha^?1 a^?1) than smallholder tea plantations (0.1 kg N₂O–N ha^?1 a^?1 and 2.1 kg NO–N ha^?1 a^?1) or grazing land (0.1 kg N₂O–N ha^?1 a^?1 and 1.1 kg NO–N ha^?1 a^?1). High soil NO fluxes were probably the consequence of long-term N fertilization and associated soil acidification, likely promoting chemodenitrification. Our experimental approach can be implemented in understudied regions, with the potential to increase the amount of information on production and consumption of trace gases from soils.     

The McClure and Weiss models of Fe–O2 bonding for oxyhemes, and the HbO2 + NO reaction

For the Fe–O₂(S = 0) linkages of oxyhemes, valence bond (VB) structures are re-presented for the McClure [Fe^II(S = 1) + O₂(S = 1)], Pauling–Coryell [Fe^II(S = 0) + O₂*(S = 0)], and Weiss [Fe^III(S = ½) + O₂ ^?(S = ½)] models of bonding. The VB structures for the McClure and Weiss models are of the increased-valence type, with more electrons participating in bonding than occur in their component Lewis structures. The Fe–O bond number and O–O bond order for the McClure structure are correlated with measured Fe–O and O–O bond lengths for oxymyoglobin. Back-bonding from O ₂ ^? to Fe^III of the Weiss structure gives a restricted form of the McClure structure. The McClure and Weiss increased-valence structures are used to provide VB formulations of mechanisms for the oxyhemoglobin + NO reaction. The products of these two formulations are Hb⁺ and NO₃ ^? (where Hb is hemoglobin) and Hb⁺ and OONO^?, respectively. Because Hb⁺ and NO₃ ^? are the observed products, they provide an experimental procedure for distinguishing the McClure and Weiss models. It is also shown that the same type of agreement between McClure-type theory and experiment occurs for oxycoboglobin + NO, cytochrome P450 monooxygenases, and related hydrogen atom transfer reactions. In the appendices, the results of density functional theory and multireference molecular orbital calculations for oxyhemes are related to one formulation of the increased-valence wavefunction for the McClure model, and theory is presented for the calculation of approximate weights for the Lewis structures that are components of the McClure increased-valence structure.     

Autotrophic denitrification by nitrate-dependent Fe(II) oxidation in a continuous up-flow biofilter

A continuous-upflow biofilter packed with sponge iron was constructed for nitrate removal under an anaerobic atmosphere. Microbacterium sp. W5, a nitrate reducing and Fe(II) oxidizing strain, was added to the biofilter as an inoculum. The best results were achieved when NO₃ ^?-N concentration was 30 mg/L and Fe²⁺ was 800 mg/L. Nitrite in influent would inhibit nitrate removal and aqueous Fe²⁺ resulted in encrustation. Fe(II)EDTA would prevent cells from encrustation and the maximum nitrogen removal efficiency was about 90 % with Fe(II)EDTA level of 1100 mg/L. Nitrate reduction followed first-order reaction kinetics. Characteristics of biofilms were analyzed by X-ray fluorescence spectroscopy.     

Cadmium(II) complex formation with glutathione

Complex formation between heavy metal ions and glutathione (GSH) is considered as the initial step in many detoxification processes in living organisms. In this study the structure and coordination between the cadmium(II) ion and GSH were investigated in aqueous solutions (pH 7.5 and 11.0) and in the solid state, using a combination of spectroscopic techniques. The similarity of the Cd K-edge and L₃-edge X-ray absorption spectra of the solid compound [Cd(GS)(GSH)]ClO₄·3H₂O, precipitating at pH 3.0, with the previously studied cysteine compound {Cd(HCys)₂·H₂O}₂·H₃O⁺·ClO₄ ^? corresponds to Cd(S–GS)₃O (dominating) and Cd(S–GS)₄ four-coordination within oligomeric complexes with mean bond distances of 2.51 ± 0.02 Å for Cd–S and 2.24 ± 0.04 Å for Cd–O. For cadmium(II) solutions (C _Cd(II) ~ 0.05 M) at pH 7.5 with moderate excess of GSH (C _GSH/C _Cd(II) = 3.0–5.0), a mix of Cd(S–GS)₃O (dominating) and Cd(S–GS)₄ species is consistent with the broad ¹¹³Cd NMR resonances in the range 632–658 ppm. In alkaline solutions (pH 11.0 and C _GSH/C _Cd(II) = 2.0 or 3.0), two distinct peaks at 322 and 674 ppm are obtained. The first peak indicates six-coordinated mononuclear and dinuclear complexes with CdS₂N₂(N/O)₂ and CdSN₃O₂ coordination in fast exchange, whereas the second corresponds to Cd(S–GS)₄ sites. At high ligand excess the tetrathiolate complex, Cd(S–GS)₄, characterized by a sharp δ(¹¹³Cd) NMR signal at 677 ppm, predominates. The average Cd–S distance, obtained from the X-ray absorption spectra, varied within a narrow range, 2.49–2.53 Å, for all solutions (pH 7.5 and 11.0) regardless of the coordination geometry.     

Spectroscopic identification of a dinuclear metal centre in manganese(II)-activated aminopeptidase P from Escherichia coli: implications for human prolidase

Electron paramagnetic resonance (EPR) spectra and X-ray absorption (EXAFS and XANES) data have been recorded for the manganese enzyme aminopeptidase P (AMPP, PepP protein) from Escherichia coli. The biological function of the protein, a tetramer of 50-kDa subunits, is the hydrolysis of N-terminal Xaa-Pro peptide bonds. Activity assays confirm that the enzyme is activated by treatment with Mn²⁺. The EPR spectrum of Mn²⁺–activated AMPP at liquid-He temperature is characteristic of an exchange-coupled dinuclear Mn(II) site, the Mn-Mn separation calculated from the zero-field splitting D of the quintet state being 3.5?(±0.1)?Å. In the X-ray absorption spectrum of Mn²⁺–activated AMPP at the Mn K edge, the near-edge features are consistent with octahedrally coordinated Mn atoms in oxidation state +2. EXAFS data, limited to k≤12?Å^–1 by traces of Fe in the protein, are consistent with a single coordination shell occupied predominantly by O donor atoms at an average Mn-ligand distance of 2.15?Å, but the possibility of a mixture of O and N donor atoms is not excluded. The Mn-Mn interaction at 3.5?Å is not detected in the EXAFS, probably due to destructive interference from light outer-shell atoms. The biological function, amino acid sequence and metal-ion dependence of E. coli AMPP are closely related to those of human prolidase, an enzyme that specifically cleaves Xaa-Pro dipeptides. Mutations that lead to human prolidase deficiency and clinical symptoms have been identified. Several known inhibitors of prolidase also inhibit AMPP. When these inhibitors are added to Mn²⁺–activated AMPP, the EPR spectrum and EXAFS remain unchanged. It can be inferred that the inhibitors either do not bind directly to the Mn centres, or substitute for existing Mn ligands without a significant change in donor atoms or coordination geometry. The conclusions from the spectroscopic measurements on AMPP have been verified by, and complement, a recent crystal structure analysis.     

Production,characteristics and applications of phytase from a rhizosphere isolated <Emphasis Type="Italic">Enterobacter</Emphasis> sp. ACSS

Optimization of process parameters for phytase production by Enterobacter sp. ACSS led to a 4.6-fold improvement in submerged fermentation, which was enhanced further in fed-batch fermentation. The purified 62 kDa monomeric phytase was optimally active at pH 2.5 and 60 °C and retained activity over a wide range of temperature (40–80 °C) and pH (2.0–6.0) with a half-life of 11.3 min at 80 °C. The kinetic parameters K _m, V _max, K _cat, and K _cat/K _m of the pure phytase were 0.21 mM, 131.58 nmol mg^?1 s^?1, 1.64 × 10³ s^?1, and 7.81 × 10⁶ M^?1 s^?1, respectively. The enzyme was fairly stable in the presence of pepsin under physiological conditions. It was stimulated by Ca⁺², Mg⁺² and Mn⁺², but inhibited by Zn⁺², Cu⁺², Fe⁺², Pb⁺², Ba⁺² and surfactants. The enzyme can be applied in dephytinizing animal feeds, and the baking industry.     

X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus

Mo K-edge X-ray absorption spectroscopy (XAS) has been used to probe the environment of Mo in dimethylsulfoxide (DMSO) reductase from Rhodobacter capsulatus in concert with protein crystallographic studies. The oxidised (Mo^VI) protein has been investigated in solution at 77?K; the Mo K-edge position (20006.4?eV) is consistent with the presence of Mo^VI and, in agreement with the protein crystallographic results, the extended X-ray absorption fine structure (EXAFS) is also consistent with a seven-coordinate site. The site is composed of one oxo-group (Mo=O 1.71?Å), four S atoms (considered to arise from the dithiolene groups of the two molybdopterins, two at 2.32?Å and two at 2.47?Å, and two O atoms, one at 1.92?Å (considered to be H-bonded to Trp 116) and one at 2.27?Å (considered to arise from Ser 147). The Mo K-edge XAS recorded for single crystals of oxidised (Mo^VI) DMSO reductase at 77?K showed a close correspondence to the data for the frozen solution but had an inferior signal:noise ratio. The dithionite-reduced form of the enzyme and a unique form of the enzyme produced by the addition of dimethylsulfide (DMS) to the oxidised (Mo^VI) enzyme have essentially identical energies for the Mo K-edge, at 20004.4?eV and 20004.5?eV, respectively; these values, together with the lack of a significant presence of Mo^V in the samples as monitored by EPR spectroscopy, are taken to indicate the presence of Mo^IV. For the dithionite-reduced sample, the Mo K-edge EXAFS indicates a coordination environment for Mo of two O atoms, one at 2.05?Å and one at 2.51?Å, and four S atoms at 2.36?Å. The coordination environment of the Mo in the DMS-reduced form of the enzyme involves three O atoms, one at 1.69?Å, one at 1.91?Å and one at 2.11?Å, plus four S atoms, two at 2.28?Å and two at 2.37?Å. The EXAFS and the protein crystallographic results for the DMS-reduced form of the enzyme are consistent with the formation of the substrate, DMSO, bound to Mo^IV with an Mo-O bond of length 1.92?Å.     

The kinetics of cyanide binding by human erythrocyte catalase

The kinetics of the binding reaction of cyanide by human erythrocyte catalase at 25 °C has been studied over the pH range 4.2 to 10.2 by means of temperature jump and stopped flow techniques. Catalase reacts with cyanide at a constant rate in the range pH 4.2 to 8.1 which decreases at higher pH. This is most simply explained by the reaction of catalase with unionized hydrogen cyanide molecules. The pH-independent rate constant for the formation of the catalase-cyanide complex is (1.3 ± 0.1) × 10⁶m^?1 s^?1. The association equilibrium constant and the dissociation rate constant for the catalase-cyanide complex were determined from the relaxation amplitudes of temperature jump experiments and by spectrophotometric titration and are (3.1 ± 0.2) × 10⁵m^?1 and 4.2 ± 0.6 s^?1, respectively in the pH-independent region.     

Effects of iron on fatty acid and astaxanthin accumulation in mixotrophic Chromochloris zofingiensis

The integration of oleaginous microalgae cultivation with high-value products is considered a low-cost approach for manufacturing algae-based biodiesel. The objective of this study was to investigate the potential of using Fe(II) to produce fatty acids and astaxanthin in mixotrophic Chromochloris zofingiensis. Fatty acid biosynthesis was less sensitive than astaxanthin formation to the changes in Fe²⁺ concentrations. However, the enhancement and inhibition of fatty acids formation were concomitant with an increase and a decrease in the production of astaxanthin, respectively. The highest contents of astaxanthin and total fatty acids were simultaneously obtained at 0.2 mM Fe²⁺ with the corresponding values of 2.2 mg g^?1 (i.e., 25.8 mg l^?1) and 41.8 % dry weight (i.e., 5 g l^?1).