Rapid loss of structural motifs in the manganese complex of oxygenic photosynthesis by X-ray irradiation at 10-300 K

Structural changes upon photoreduction caused by x-ray irradiation of the water-oxidizing tetramanganese complex of photosystem II were investigated by x-ray absorption spectroscopy at the manganese K-edge. Photoreduction was directly proportional to the x-ray dose. It was faster in the higher oxidized S2 state than in S1; seemingly the oxidizing potential of the metal site governs the rate. X-ray irradiation of the S1 state at 15 K initially caused single-electron reduction to S0* accompanied by the conversion of one di-mu-oxo bridge between manganese atoms, previously separated by approximately 2.7 A, to a mono-mu-oxo motif. Thereafter, manganese photoreduction was 100 times slower, and the biphasic increase in its rate between 10 and 300 K with a breakpoint at approximately 200 K suggests that protein dynamics is rate-limiting the radical chemistry. For photoreduction at similar x-ray doses as applied in protein crystallography, halfway to the final Mn(II)4 state the complete loss of inter-manganese distances <3 A was observed, even at 10 K, because of the destruction of mu-oxo bridges between manganese ions. These results put into question some structural attributions from recent protein crystallography data on photosystem II. It is proposed to employ controlled x-ray photoreduction in metalloprotein research for: (i) population of distinct reduced states, (ii) estimating the redox potential of buried metal centers, and (iii) research on protein dynamics.     

Fate of oxygen species from O2 activation at dimetal cofactors in an oxidase enzyme revealed by 57Fe nuclear resonance X-ray scattering and quantum chemistry

Oxygen (O₂) activation is a central challenge in chemistry and catalyzed at prototypic dimetal cofactors in biological enzymes with diverse functions. Analysis of intermediates is required to elucidate the reaction paths of reductive O₂ cleavage. An oxidase protein from the bacterium Geobacillus kaustophilus, R2lox, was used for aerobic in-vitro reconstitution with only ⁵⁷Fe(II) or Mn(II) plus ⁵⁷Fe(II) ions to yield [FeFe] or [MnFe] cofactors under various oxygen and solvent isotopic conditions including ^16/18O and H/D exchange. ⁵⁷Fe-specific X-ray scattering techniques were employed to collect nuclear forward scattering (NFS) and nuclear resonance vibrational spectroscopy (NRVS) data of the R2lox proteins. NFS revealed Fe/Mn(III)Fe(III) cofactor states and Mössbauer quadrupole splitting energies. Quantum chemical calculations of NRVS spectra assigned molecular structures, vibrational modes, and protonation patterns of the cofactors, featuring a terminal water (H₂O) bound at iron or manganese in site 1 and a metal-bridging hydroxide (μOH⁻) ligand. A procedure for quantitation and correlation of experimental and computational NRVS difference signals due to isotope labeling was developed. This approach revealed that the protons of the ligands as well as the terminal water at the R2lox cofactors exchange with the bulk solvent whereas ¹⁸O from ¹⁸O₂ cleavage is incorporated in the hydroxide bridge. In R2lox, the two water molecules from four-electron O₂ reduction are released in a two-step reaction to the solvent. These results establish combined NRVS and QM/MM for tracking of iron-based oxygen activation in biological and chemical catalysts and clarify the reductive O₂ cleavage route in an enzyme.     

High-valent [MnFe] and [FeFe] cofactors in ribonucleotide reductases

Ribonucleotide reductases (RNRs) are essential for DNA synthesis in most organisms. In class-Ic RNR from Chlamydia trachomatis (Ct), a MnFe cofactor in subunit R2 forms the site required for enzyme activity, instead of an FeFe cofactor plus a redox-active tyrosine in class-Ia RNRs, for example in mouse (Mus musculus, Mm). For R2 proteins from Ct and Mm, either grown in the presence of, or reconstituted with Mn and Fe ions, structural and electronic properties of higher valence MnFe and FeFe sites were determined by X-ray absorption spectroscopy and complementary techniques, in combination with bond-valence-sum and density functional theory calculations. At least ten different cofactor species could be tentatively distinguished. In Ct R2, two different Mn(IV)Fe(III) site configurations were assigned either L(4)Mn(IV)(μO)(2)Fe(III)L(4) (metal-metal distance of ~2.75?, L = ligand) prevailing in metal-grown R2, or L(4)Mn(IV)(μO)(μOH)Fe(III)L(4) (~2.90?) dominating in metal-reconstituted R2. Specific spectroscopic features were attributed to an Fe(IV)Fe(III) site (~2.55?) with a L(4)Fe(IV)(μO)(2)Fe(III)L(3) core structure. Several Mn,Fe(III)Fe(III) (~2.9-3.1?) and Mn,Fe(III)Fe(II) species (~3.3-3.4?) likely showed 5-coordinated Mn(III) or Fe(III). Rapid X-ray photoreduction of iron and shorter metal-metal distances in the high-valent states suggested radiation-induced modifications in most crystal structures of R2. The actual configuration of the MnFe and FeFe cofactors seems to depend on assembly sequences, bound metal type, valence state, and previous catalytic activity involving subunit R1. In Ct R2, the protonation of a bridging oxide in the Mn(IV)(μO)(μOH)Fe(III) core may be important for preventing premature site reduction and initiation of the radical chemistry in R1.     

A mixed valence form of the iron cluster in the B2 protein of ribonucleotide reductase from Escherichia coli.

A mixed valent form of the iron cluster (Fe(II)Fe(III) in the B2 protein of ribonucleotide reductase has been isolated and characterized. The irons in this state of the protein are ferromagnetically coupled as indicated by the observation of a novel S = 9/2 EPR spectrum. This is the first ferromagnetically coupled Fe(II)Fe(III) cluster reported for a protein and the first observation of the mixed valence form of ribonucleotide reductase.     

Mono- and dinuclear Fe(III) complexes with the N2O2 donor 5-chlorosalicylideneimine ligands; synthesis, X-ray structural characterization and magnetic properties

Two novel monomeric [C₁₈H₁₇Cl₃N₂O₂Fe] (1) and dimeric [C₃₈H₃₆N₄O₄Cl₆Fe₂] (2) Fe(III) tetradentate Schiff base complexes have been synthesized and their crystal structures have been determined by single crystal X-ray diffraction analysis. In complex (1) the Schiff base ligand coordinates toward one iron atom in a tetradentate mode and each iron atom is five coordinated with the coordination geometry around iron atom which can be described as a distorted square pyramid. The presence of a short (2.89 Å) non-bonding interatomic Fe···O distances between adjacent monomeric Fe(III) complexes results in the formation of a dimer. Structural analysis of compound (2) shows that the structure is a centrosymmetric dimer in which the six coordinated Fe(III) atoms are linked by μ-phenoxo bridges from one of the phenolic oxygen atoms of each Schiff base ligand to the opposite metal center. The variable-temperature (2-300 K) magnetic susceptibility (χ) data of these two compounds have been investigated. The results show that for both complexes Fe(III) centers are in the high spin configuration (S = 5/2) and indicate antiferromagnetic spin-exchange interaction between Fe(III) ions. The obtained results are briefly discussed using magnetostructural correlations developed for other class of iron(III) complexes.     

Oxygen cleavage with manganese and iron in ribonucleotide reductase from <Emphasis Type="Italic">Chlamydia trachomatis</Emphasis>

The oxygen cleavage in Chlamydia trachomatis ribonucleotide reductase (RNR) has been studied using B3LYP* hybrid density functional theory. Class Ic C. trachomatis RNR lacks the radical-bearing tyrosine, crucial for activity in conventional class I (subclass a and b) RNR. Instead of the Fe(III)Fe(III)–Tyr(rad) active state, C. trachomatis RNR has a mixed Mn(IV)Fe(III) metal center in subunit II (R2). A mixed MnFe metal center has never been observed as a radical cofactor before. The active state is generated by reductive oxygen cleavage at the metal site. On the basis of calculated barriers for oxygen cleavage in C. trachomatis R2 and R2 from Escherichia coli with a diiron, a mixed manganese–iron, and a dimanganese center, conclusions can be drawn about the effect of changing metals in R2. The oxygen cleavage is found to be governed by two factors: the redox potentials of the metals and the relative stability of the different peroxides. Mn(IV) has higher stability than Fe(IV), and the barrier is therefore lower with a mixed metal center than with a diiron center. With a dimanganese center, an asymmetric peroxide is more stable than the symmetric peroxide, and the barrier therefore becomes too high. Calculated proton-coupled redox potentials are compared to identify three possible R2 active states, the Fe(III)Fe(III)–Tyr(rad) state, the Mn(IV)Fe(III) state, and the Mn(IV)Mn(IV) state. A tentative energy profile of the thermodynamics of the radical transfer from R2 to subunit I is constructed to illustrate how the stability of the active states can be understood from a thermodynamical point of view.     

X-ray reduction correlates with soaking accessibility as judged from four non-crystallographically related diiron sites

X-ray crystallography is extensively used to determine the atomic structure of proteins and their cofactors. Though a commonly overlooked problem, it has been shown that structural damage to a redox active metal site may precede loss of diffractivity by more than an order of magnitude in X-ray dose. Therefore the risk of misassigning redox states is great. Adequate treatment and consideration of this issue is of paramount importance in metalloprotein science, from experimental design to interpretation of the data and results. Some metal sites appear to be much more amenable to reduction than others, but the underlying processes are poorly understood. Here, we have analyzed the four non-crystallographically related diiron sites in a crystal of the ribonucleotide reductase R2F protein from Corynebacterium ammoniagenes. We conclude that the amount of X-ray reduction a metal site suffers correlates with its soaking accessibility. This direct observation supports the hypothesis that a diffusion component is involved in the X-ray reduction process.     

Tyrosyl free radical formation in the small subunit of mouse ribonucleotide reductase

Each R2 subunit of mammalian ribonucleotide reductase contains a pair of high spin ferric ions and a tyrosyl free radical essential for activity. To study the mechanism of tyrosyl radical formation, substoichiometric amounts of Fe(II) were added to recombinant mouse R2 apoprotein under strictly anaerobic conditions and then the solution was exposed to air. Low temperature EPR spectroscopy showed that the signal from the generated tyrosyl free radical correlated well with the quantity of the Fe(II) added with a stoichiometry of 3 Fe(II) needed to produce 1 tyrosyl radical: 3 Fe(II) + P + O2 + Tyr-OH + H+----Fe(III)O2-Fe(III)-P + H2O. + Tyr-O. + Fe(III), where P is an iron-binding site of protein R2 and Tyr-OH is the active tyrosyl residue. The O-O bond of a postulated intermediate O2(2-)-Fe(III)2-P state is cleaved by the extra electron provided by Fe(II) leading to formation of OH., which in turn reacts with Tyr-OH to give Tyr-O.. In the presence of ascorbate, added to reduce the monomeric Fe(III) formed, 80% of the Fe(II) added produced a radical. The results strongly indicate that each dimeric Fe(III) center during its formation can generate a tyrosyl-free radical and that iron binding to R2 apoprotein is highly cooperative.     

Structural studies on a mitochondrial glyoxalase II

Glyoxalase 2 is a beta-lactamase fold-containing enzyme that appears to be involved with cellular chemical detoxification. Although the cytoplasmic isozyme has been characterized from several organisms, essentially nothing is known about the mitochondrial proteins. As a first step in understanding the structure and function of mitochondrial glyoxalase 2 enzymes, a mitochondrial isozyme (GLX2-5) from Arabidopsis thaliana was cloned, overexpressed, purified, and characterized using metal analyses, EPR and (1)H NMR spectroscopies, and x-ray crystallography. The recombinant enzyme was shown to bind 1.04 +/- 0.15 eq of iron and 1.31 +/- 0.05 eq of Zn(II) and to exhibit k(cat) and K(m) values of 129 +/- 10 s(-1) and 391 +/- 48 microm, respectively, when using S-d-lactoylglutathione as the substrate. EPR spectra revealed that recombinant GLX2-5 contains multiple metal centers, including a predominant Fe(III)Z-n(II) center and an anti-ferromagnetically coupled Fe(III)Fe(II) center. Unlike cytosolic glyoxalase 2 from A. thaliana, GLX2-5 does not appear to specifically bind manganese. (1)H NMR spectra revealed the presence of at least eight paramagnetically shifted resonances that arise from protons in close proximity to a Fe(III)Fe(II) center. Five of these resonances arose from solvent-exchangeable protons, and four of these have been assigned to NH protons on metal-bound histidines. A 1.74-A resolution crystal structure of the enzyme revealed that although GLX2-5 shares a number of structural features with human GLX2, several important differences exist. These data demonstrate that mitochondrial glyoxalase 2 can accommodate a number of different metal centers and that the predominant metal center is Fe(III)Zn(II).     

Ferric Ion and Oxygen Reduction at the Surface of Protoplasts and Cells of Acer pseudoplatanus

This work was undertaken to verify whether surface NADH oxidases or peroxidases are involved in the apoplastic reduction of Fe(III). The reduction of Fe(III)-ADP, linked to NADH-dependent activity of horseradish peroxidase (HRP), protoplasts and cells of Acer pseudoplatanus, was measured as Fe(II)-bathophenanthrolinedisulfonate (BPDS) chelate formation. In the presence of BPDS in the incubation medium (method 1), NADH-dependent HRP activity was associated with a rapid Fe(III)-ADP reduction that was almost completely inhibited by superoxide dismutase (SOD), while catalase only slowed down the rate of reduction. A. pseudoplatanus protoplasts and cells reduced extracellular Fe(III)-ADP in the absence of exogenously supplied NADH. The addition of NADH stimulated the reduction. SOD and catalase only inhibited the NADH-dependent Fe(III)-ADP reduction. Mn(II), known for its ability to scavenge O^?₂, inhibited both the independent and NADH-dependent Fe(III)-ADP reduction. The reductase activity of protoplasts and cells was also monitored in the absence of BPDS (method 2). The latter was added only at the end of the reaction to evaluate Fe(II) formed. Also, in this case, both preparations reduced Fe(III)-ADP. However, the addition of NADH did not stimulate Fe(III)-ADP reduction but, instead, lowered it. This may be related to a re-oxidation of Fe(II) by H₂O₂ that could also be produced during NADH-dependent peroxidase activity. Catalase and SOD made the Fe(III)-ADP reduction more efficient because, by removing H₂O₂ (catalase) or preventing H₂O₂ formation (SOD), they hindered the re-oxidation of Fe(II) not chelated by BPDS. As with the result obtained by method 1, Mn(II) inhibited Fe(III)-ADP reduction carried out in the presence or absence of NADH. The different effects of SOD and Mn(II), both scavengers of O^?₂, may depend on the ability of Mn(II) to permeate the cells more easily than SOD. These results show that A. pseudoplatanus protoplasts and cells reduce extracellular Fe(III)-ADP. Exogenously supplied NADH induces an additional reduction of Fe(III) by the activity of NADH peroxidases of the plasmalemma or cell wall. However, the latter can also trigger the formation of H₂O₂ that, reacting with Fe(II) (not chelated by BPDS), generates hydroxyl radicals and converts Fe(II) to Fe(III) (Fenton's reaction).     

Structural characterization of a metal-based perfusion tracer: Copper(II) pyruvaldehyde bis (N4-methylthiosemicarbazone)

Copper(II) pyruvaldehyde bis(N⁴-methylthiosemicarbazone), Cu(PTSM), has been obtained as a dark red crystalline solid from EtOH-DMSO solvent mixture and structurally characterized by x-ray crystallography. The molecule possesses the expected pseudo-square planar N₂S₂ metal coordination sphere; however, the copper center also interacts through its axial coordination site with the sulfur atom of an adjacent Cu(PTSM) molecule in the crystal lattice. The structure of this compound is compared with the structures of other metal complexes that have been proposed in the nuclear medicine literature as perfusion tracers.     

Orientation of the tyrosyl radical in Salmonella typhimurium class Ib ribonucleotide reductase determined by high field EPR of R2F single crystals

The R2 protein of class I ribonucleotide reductase (RNR) generates and stores a tyrosyl radical, located next to a diferric iron center, which is essential for ribonucleotide reduction and thus DNA synthesis. X-ray structures of class Ia and Ib proteins from various organisms served as bases for detailed mechanistic suggestions. The active site tyrosine in R2F of class Ib RNR of Salmonella typhimurium is located at larger distance to the diiron site, and shows a different side chain orientation, as compared with the tyrosine in R2 of class Ia RNR from Escherichia coli.No structural information has been available for the active tyrosyl radical in R2F. Here we report on high field EPR experiments of single crystals of R2F from S. typhimurium, containing the radical Tyr-105*. Full rotational pattern of the spectra were recorded, and the orientation of the g-tensor axes were determined, which directly reflect the orientation of the radical Tyr-105* in the crystal frame. Comparison with the orientation of the reduced tyrosine Tyr-105-OH from the x-ray structure reveals a rotation of the tyrosyl side chain, which reduces the distance between the tyrosyl radical and the nearest iron ligands toward similar values as observed earlier for Tyr-122* in E. coli R2. Presence of the substrate binding subunit R1E did not change the EPR spectra of Tyr-105*, indicating that binding of R2E alone induces no structural change of the diiron site. The present study demonstrates that structural and functional information about active radical states can be obtained by combining x-ray and high-field-EPR crystallography.     

Regulation of Dissimilatory Fe(III) Reduction Activity in Shewanella putrefaciens

Under anaerobic conditions, Shewanella putrefaciens is capable of respiratory-chain-linked, high-rate dissimilatory iron reduction via both a constitutive and inducible Fe(III)-reducing system. In the presence of low levels of dissolved oxygen, however, iron reduction by this microorganism is extremely slow. Fe(II)-trapping experiments in which Fe(III) and O₂ were presented simultaneously to batch cultures of S. putrefaciens indicated that autoxidation of Fe(II) was not responsible for the absence of Fe(III) reduction. Inhibition of cytochrome oxidase with CN⁻ resulted in a high rate of Fe(III) reduction in the presence of dissolved O₂, which suggested that respiratory control mechanisms did not involve inhibition of Fe(III) reductase activities or Fe(III) transport by molecular oxygen. Decreasing the intracellular ATP concentrations by using an uncoupler, 2,4-dinitrophenol, did not increase Fe(III) reduction, indicating that the reduction rate was not controlled by the energy status of the cell. Control of electron transport at branch points could account for the observed pattern of respiration in the presence of the competing electron acceptors Fe(III) and O₂.     

Conversion of a heme-based oxygen sensor to a heme oxygenase by hydrogen sulfide: effects of mutations in the heme distal side of a heme-based oxygen sensor phosphodiesterase (Ec DOS)

The heme-based oxygen-sensor phosphodiesterase from Escherichia coli (Ec DOS), is composed of an N-terminal heme-bound oxygen sensing domain and a C-terminal catalytic domain. Oxygen (O₂) binding to the heme Fe(II) complex in Ec DOS substantially enhances catalysis. Addition of hydrogen sulfide (H₂S) to the heme Fe(III) complex in Ec DOS also remarkably stimulates catalysis in part due to the heme Fe(III)–SH and heme Fe(II)–O₂ complexes formed by H₂S. In this study, we examined the roles of the heme distal amino acids, M95 (the axial ligand of the heme Fe(II) complex) and R97 (the O₂ binding site in the heme Fe(II)–O₂ complex) of the isolated heme-binding domain of Ec DOS (Ec DOS-PAS) in the binding of H₂S under aerobic conditions. Interestingly, R97A and R97I mutant proteins formed an oxygen-incorporated modified heme, verdoheme, following addition of H₂S combined with H₂O₂ generated by the reactions. Time-dependent mass spectroscopic data corroborated the findings. In contrast, H₂S did not interact with the heme Fe(III) complex of M95H and R97E mutants. Thus, M95 and/or R97 on the heme distal side in Ec DOS-PAS significantly contribute to the interaction of H₂S with the Fe(III) heme complex and also to the modification of the heme Fe(III) complex with reactive oxygen species. Importantly, mutations of the O₂ binding site of the heme protein converted its function from oxygen sensor to that of a heme oxygenase. This study establishes the novel role of H₂S in modifying the heme iron complex to form verdoheme with the aid of reactive oxygen species.     

Iron complexes of chiral phenol-oxazoline ligands: Structural studies and oxidation catalysis

Iron complexes of two ligands, HphoxCOOH and HphoxiPr, have been synthesized and characterized by crystal structure analyses. The complexes (HNEt₃)₂[Fe(phoxCOO)₂](ClO₄) and [Fe(phoxiPr)₃] are reported. Reactions of the ligands rac-HphoxCOOH and rac-HphoxiPr with iron(II) or iron(III) perchlorate result in the formation of iron(III) complexes with pseudo-octahedral geometry around the metal center. The iron complex obtained from rac-HphoxCOOH crystallized in the centrosymmetric space group Cmca. The two ligands are bound in a tridentate manner generating a meridional coordination with both dianionic ligands on a metal center having the same chirality; due to the center of symmetry the complex with opposite chirality is also present. The complex (HNEt₃)₂[Fe(phoxCOO)₂](ClO₄) is the first accurate structural model of the iron complex of a siderophore analog commonly observed in mycobactins. The three didentate ligands in the complex [Fe(phoxiPr)₃] are bound with like atoms in a meridional manner to the metal center. The metal ion is surrounded by two ligands of the same chirality and one ligand of opposite chirality (ie. RRS or SSR); due to the presence of a center of symmetry both isomers are present in the crystal structure. The complex (HNEt₃)₂[Fe(phoxCOO)₂](ClO₄) shows promising activity in the oxidation of alkanes, such as toluene, ethylbenzene and cumene, while the complex [Fe(phoxiPr)₃] does not show any catalytic activity in alkane oxidations under the conditions tested. The complex (HNEt₃)₂[Fe(phoxCOO)₂](ClO₄) is reasonably efficient in the conversion of H₂O₂ to oxidation products.     

Induction of iron(III) and copper(II) reduction in pea (Pisum sativum L.) roots by Fe and Cu status: Does the root-cell plasmalemma Fe(III)-chelate reductase perform a general role in regulating cation uptake?

We investigated the effects of Fe and Cu status of pea (Pisum sativum L.) seedlings on the regulation of the putative root plasma-membrane Fe(III)-chelate reductase that is involved in Fe(III)-chelate reduction and Fe²⁺ absorption in dicotyledons and nongraminaceous monocotyledons. Additionally, we investigated the ability of this reductase system to reduce Cu(II)-chelates as well as Fe(III)-chelates. Pea seedlings were grown in full nutrient solutions under control, -Fe, and -Cu conditions for up to 18 d. Iron(III) and Cu(II) reductase activity was visualized by placing roots in an agarose gel containing either Fe(III)-EDTA and the Fe(II) chelate, Na₂bathophenanthrolinedisulfonic acid (BPDS), for Fe(III) reduction, or CuSO₄, Na₃citrate, and Na₂-2,9-dimethyl-4,7-diphenyl-1, 10-phenanthrolinedisulfonic acid (BCDS) for Cu(II) reduction. Rates of root Fe(III) and Cu(II) reduction were determined via spectrophotometric assay of the Fe(II)-BPDS or the Cu(I)-BCDS chromophore. Reductase activity was induced or stimulated by either Fe deficiency or Cu depletion of the seedlings. Roots from both Fe-deficient and Cu-depleted plants were able to reduce exogenous Cu(II)-chelate as well as Fe(III)-chelate. When this reductase was induced by Fe deficiency, the accumulation of a number of mineral cations (i.e., Cu, Mn, Fe, Mg, and K) in leaves of pea seedlings was significantly increased. We suggest that, in addition to playing a critical role in Fe absorption, this plasma-membrane reductase system also plays a more general role in the regulation of cation absorption by root cells, possibly via the reduction of critical sulfhydryl groups in transport proteins involved in divalent-cation transport (divalent-cation channels?) across the root-cell plasmalemma.     

Structural insights into the enzyme catalysis from comparison of three forms of dissimilatory sulphite reductase from Desulfovibrio gigas

The crystal structures of two active forms of dissimilatory sulphite reductase (Dsr) from Desulfovibrio gigas, Dsr‐I and Dsr‐II, are compared at 1.76 and 2.05 Å resolution respectively. The dimeric α₂β₂γ₂ structure of Dsr‐I contains eight [4Fe–4S] clusters, two saddle‐shaped sirohaems and two flat sirohydrochlorins. In Dsr‐II, the [4Fe–4S] cluster associated with the sirohaem in Dsr‐I is replaced by a [3Fe–4S] cluster. Electron paramagnetic resonance (EPR) of the active Dsr‐I and Dsr‐II confirm the co‐factor structures, whereas EPR of a third but inactive form, Dsr‐III, suggests that the sirohaem has been demetallated in addition to its associated [4Fe–4S] cluster replaced by a [3Fe–4S] centre. In Dsr‐I and Dsr‐II, the sirohydrochlorin is located in a putative substrate channel connected to the sirohaem. The γ‐subunit C‐terminus is inserted into a positively charged channel formed between the α‐ and β‐subunits, with its conserved terminal Cysγ104 side‐chain covalently linked to the CHA atom of the sirohaem in Dsr‐I. In Dsr‐II, the thioether bond is broken, and the Cysγ104 side‐chain moves closer to the bound sulphite at the sirohaem pocket. These different forms of Dsr offer structural insights into a mechanism of sulphite reduction that can lead to S₃O₆^2?, S₂O₃^2? and S^2?.     

Histidine ligand variants of a flavo-diiron protein: effects on structure and activities

Flavo-diiron proteins (FDPs) contain non-heme diiron and proximal flavin mononucleotide (FMN) active sites and function as terminal components of a nitric oxide reductase (NOR) and/or a four-electron dioxygen reductase (O₂R). While most FDPs show similar structural, spectroscopic, and redox properties, O₂R and NOR activities vary significantly among FDPs. A potential source of this variability is the iron ligation status of a conserved His residue that provides an iron ligand in all known FDP structures but one, where this His residue is rotated away from iron and replaced by a solvent ligand. In order to test the effect of this His ligation status, we changed this ligating His residue (H90) in Thermotoga maritima (Tm) FDP to either Asn or Ala. The wild-type Tm FDP shows significantly higher O₂R than NOR activity. Single crystal X-ray crystallography revealed a remarkably conserved diiron site structure in the H90N and ?A variants, differing mainly by either Asn or solvent coordination, respectively, in place of H90. The steady-state activities were minimally affected by the H90 substitutions, remaining significantly higher for O₂R versus NOR. The pre-steady-state kinetics of the fully reduced FDP with O₂ were also minimally affected by the H90 substitutions. The results indicate that the coordination status of this His ligand does not significantly modulate the O₂R or NOR activities, and that FDPs can retain these activities when the individual iron centers are differentiated by His ligand substitution. This differentiation may have implications for the O₂R and NOR mechanisms of FDPs.     

A chelate complex‐enhanced luminol system for selective determination of Co(II), Fe(II) and Cr(III)

A determination method for Co(II), Fe(II) and Cr(III) ions by luminol‐H₂O₂ system using chelating reagents is presented. A metal ion‐chelating ligand complex with a Co(II) ion and a chelating reagent like ethylenediaminetetraacetic acid (EDTA) produced highly enhanced chemiluminescence (CL) intensity as well as longer lifetime in the luminol‐H₂O₂ system compared to metals that exist as free ions. Whereas free Cu(II) and Pb(II) ions had a strong catalytic effect on the luminol‐H₂O₂ system, significantly, the complexes of Cu(II) and Pb(II) with chelating reagents lost their catalytic activity due to the chelating reagents acting as masking agents. Based on the observed phenomenon, it was possible to determine Co(II), Fe(II) and Cr(III) ions with enhanced sensitivity and selectivity using the chelating reagents of the luminol‐H₂O₂ system. The effects of ligand, H₂O₂ concentration, pH, buffer solution and concentrations of chelating reagents on CL intensity of the luminol‐H₂O₂ system were investigated and optimized for the determination of Co(II), Fe(II) and Cr(III) ions. Under optimized conditions, the calibration curve of metal ions was linear over the range of 2.0 × 10^‐8 to 2.0 × 10^‐5 M for Co(II), 1.0 × 10^‐7 to 2.0 × 10^‐5 M for Fe (II) and 2.0 × 10^‐7 to 1.0 × 10^‐4 M for Cr(III). Limits of detection (3σ/s) were 1.2 × 10^‐8, 4.0 × 10^‐8 and 1.2 × 10^‐7 M for Co(II), Fe(II) and Cr(III), respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Structure/function correlations over binuclear non-heme iron active sites

Binuclear non-heme iron enzymes activate O₂ to perform diverse chemistries. Three different structural mechanisms of O₂ binding to a coupled binuclear iron site have been identified utilizing variable-temperature, variable-field magnetic circular dichroism spectroscopy (VTVH MCD). For the μ-OH-bridged Fe(II)₂ site in hemerythrin, O₂ binds terminally to a five-coordinate Fe(II) center as hydroperoxide with the proton deriving from the μ-OH bridge and the second electron transferring through the resulting μ-oxo superexchange pathway from the second coordinatively saturated Fe(II) center in a proton-coupled electron transfer process. For carboxylate-only-bridged Fe(II)₂ sites, O₂ binding as a bridged peroxide requires both Fe(II) centers to be coordinatively unsaturated and has good frontier orbital overlap with the two orthogonal O₂ π* orbitals to form peroxo-bridged Fe(III)₂ intermediates. Alternatively, carboxylate-only-bridged Fe(II)₂ sites with only a single open coordination position on an Fe(II) enable the one-electron formation of Fe(III)–O₂ ^? or Fe(III)–NO^? species. Finally, for the peroxo-bridged Fe(III)₂ intermediates, further activation is necessary for their reactivities in one-electron reduction and electrophilic aromatic substitution, and a strategy consistent with existing spectral data is discussed.