Rhombic Species Research Articles

Synthesis, Spectroscopy, and Structure of [FeRu(μ-dithiolate)(CN)2(CO)4]2.

The salt [K(18-crown-6)]2[Ru(CN)2(CO)3] ([K(18-crown-6)]2[1]) was generated by the reaction of Ru(C2H4)(CO)4 with [K(18-crown-6)]CN. An initial thermal reaction gives [Ru(CN)(CO)4]-, which, upon ultraviolet (UV) irradiation, reacts with a second equiv of CN-. Protonation of [1]2- gave [HRu(CN)2(CO)3]- ([H1]-), which was isolated as a single isomer with mutually trans cyanide ligands. The complex cis,cis,cis-[Ru(pdt)(CN)2(CO)2]2- ([2]2-) was prepared by the UV-induced reaction of [1]2- with propanedithiol (pdtH2). The corresponding iron complex cis,cis,cis-[Fe(pdt)(CN)2(CO)2]2- ([3]2-) was prepared similarly. The pdt complexes [2]2- and [3]2- were treated with Fe(benzylideneacetone)(CO)3 to give, respectively, [RuFe (μ-pdt)(CN)2(CO)4]2- ([5]2-) and [Fe2(μ-pdt)(CN)2(CO)4]2- ([4]2-). The pathway from [3]2- to Fe2 complex [4]2- implicates intermetallic migration of CN-. In contrast, the formation of [5]2- leaves the Ru(CN)2(CO) center intact, as confirmed by X-ray crystallography. The structure of [5]2- features a "rotated" square-pyramidal Fe(CO)2(μ-CO) site. NMR measurements indicate that the octahedral Ru site is stereochemically rigid, whereas the Fe site dynamically undergoes turnstile rotation. 57Fe Mössbauer spectral parameters are very similar for rotated [5]2- and unrotated Fe2 complex [4]2-, indicating the insensitivity of that technique to both the geometry and the oxidation state of the Fe site. According to cyclic voltammetry, [5]2- oxidizes at E1/2 ∼ -0.8 V vs Fc+/0. Electron paramagnetic resonance (EPR) measurements show that 1e- oxidation of [5]2- gives an S = 1/2 rhombic species, consistent with the formulation Ru(II)Fe(I), related to the Hox state of the [FeFe] hydrogenases. Density functional theory (DFT) studies reproduce the structure, 1H NMR shifts, and infrared (IR) spectra observed for [5]2-. Related homometallic complexes with both cyanides on a single metal are predicted to not adopt rotated structures. These data suggest that [5]2- is best described as Ru(II)Fe(0). This conclusion raises the possibility that for some reduced states of the [FeFe]-hydrogenases, the [2Fe]H site may be better described as Fe(II)Fe(0) than Fe(I)Fe(I).

Boron-based inorganic heterocyclic clusters: electronic structure, chemical bonding, aromaticity, and analogy to hydrocarbons.

This Perspective article deals with recent computational and experimental findings in boron-based heterocyclic clusters, which focuses on binary B-O and B-S clusters, as well as relevant ternary B-X-H (X = O, S, N) species. Boron is electron-deficient and boron clusters do not form monocyclic rings or linear chains. Boron-based heterocyclic clusters are intuitively even more electron-deficient and feature exotic chemical bonding, which make use of O 2p, S 3p, or N 2p lone-pairs for π delocalization over heterocyclic rings, facilitating new cluster structures and new types of bonding. Rhombic, pentagonal, hexagonal, and polycyclic clusters are discussed herein. Rhombic species are stabilized by four-center four-electron (4c-4e) π bonding, that is, the o-bond. An o-bond cluster differs from a typical 4π antiaromatic system, because it has 4π electrons in an unusual bonding/nonbonding combination, which takes advantage of the empty 2pz atomic orbitals from electron-deficient boron centers. A variety of examples (notably including boronyl boroxine) possess a hexagonal ring, as well as magic 6π electron-counting, making them new members of the inorganic benzene family. Pentagonal clusters bridge rhombic o-bond systems and inorganic benzenes, but they do not necessarily favor 6π electron-counting as in cyclopentadienide anion. In contrast, pentagonal 4π clusters are stable, leading to the concept of pentagonal o-bond. One electron can overturn the potential energy landscape of a system, enabling rhombic-to-hexagonal structural transition, which further reinforces the idea that 4π electron-counting is favorable for rhombic systems and 6π is magic for hexagonal rings. The bonding analogy between heterocyclic clusters and hydrocarbons goes beyond monocyclic species, which allows rational design of boron-based inorganic analogs of polycyclic aromatic hydrocarbons, including s-indacene as a puzzling aromatic/antiaromatic system. Selected linear B-O clusters are also briefly discussed, featuring dual 3c-4e π bonds, that is, ω-hyperbonds. Dual ω-hyperbonds, rhombic or pentagonal o-bond, and inorganic benzenes share a common chemical origin. The field of boron-based heterocyclic clusters is still in its infant stage, and much new chemistry remains to be discovered in forthcoming experimental and theoretical studies.

A natural heme-signature variant of CYP267A1 from Sorangiumcellulosum So ce56 executes diverse ω-hydroxylation.

A novel naturally occurring heme-signature variant of CYP267A1 from myxobacterium Sorangiumcellulosum So ce56 and its mutant L366F, the actual mimic of the 'conserved' heme-signature of cytochromes P450, were heterologously expressed in Escherichiacoli in a soluble form and purified. The UV-visible characteristics of both variants were highly similar. Although leucine replaced the phenylalanine in the heme-signature domain of CYP267A1, EPR measurements of the ligand-free wild-type CYP267A1 and the mutant L366F showed low-spin rhombic species suggesting a conserved heme environment of the P450s. The need of primary redox partners for the orphan P450 was sustained by the bovine redox system and a class-I electron transfer path was provided during fatty acid hydroxylation. CYP267A1 showed higher activity and produced more diverse ω-hydroxylated products compared with L366F. In both enzymes the regioselectivity of the fatty acid hydroxylation shifted towards the inner carbon atoms of the fatty acid chains with increasing carbon chain lengths. Our docking results in a homology model of the protein showed that longer fatty acids need to be folded to fit into the binding pocket. In the mutant L366F, the ω-1 and ω-2 positions which exhibit the largest electron density of the highest occupied molecular orbital are preferred. It is speculated that the leucine heme-signature variant of P450 might have evolved under selective evolutionary pressure, which confers an increased advantage to generate a broader spectrum of related alcohols and carboxylic acids required for the bacterial homeostasis or metabolism in a particular ecological niche.

The c i / b H moiety in the b 6 f complex studied by EPR: A pair of strongly interacting hemes

X-band EPR features in the region of 90-150 mT have previously been attributed to heme ci of the b6 complex [Zhang H, Primak A, Bowman MK, Kramer DM, Cramer WA (2004) Biochemistry 43:16329-16336] and interpreted as arising from a high-spin species. However, the complexity of the observed spectrum is rather untypical for high-spin hemes. In this work, we show that addition of the inhibitor 2-n-nonyl-4-hydroxyquinoline N-oxide largely simplifies heme ci's EPR properties. The spectrum in the presence of 2-n-nonyl-4-hydroxyquinoline N-oxide is demonstrated to be caused by a simple S = 5/2, rhombic species split by magnetic dipolar interaction (A(xx )= 7.5 mT) with neighboring heme bH. The large spacing of lines in the uninhibited system, by contrast, cannot be rationalized solely on the basis of magnetic dipolar coupling but is likely to encompass strong contributions from exchange interactions. The role of the H2O/OH- molecule bridging heme ci's Fe atom and heme bH's propionate side chain in mediating these interactions is discussed.

Conformational Mobility in the Active Site of a Heme Peroxidase

Conformational mobility of the distal histidine residue has been implicated for several different heme peroxidase enzymes, but unambiguous structural evidence is not available. In this work, we present mechanistic, spectroscopic, and structural evidence for peroxide- and ligand-induced conformational mobility of the distal histidine residue (His-42) in a site-directed variant of ascorbate peroxidase (W41A). In this variant, His-42 binds "on" to the heme in the oxidized form, duplicating the active site structure of the cytochromes b but, in contrast to the cytochromes b, is able to swing "off" the iron during catalysis. This conformational flexibility between the on and off forms is fully reversible and is used as a means to overcome the inherently unreactive nature of the on form toward peroxide, so that essentially complete catalytic activity is maintained. Contrary to the widely adopted view of heme enzyme catalysis, these data indicate that strong coordination of the distal histidine to the heme iron does not automatically undermine catalytic activity. The data add a new dimension to our wider appreciation of structure/activity correlations in other heme enzymes.

Low‐Temperature EPR and Mössbauer Spectroscopy of Two Cytochromes with His–Met Axial Coordination Exhibiting HALS Signals

C-type cytochromes with histidine-methionine (His-Met) iron coordination play important roles in electron-transfer reactions and in enzymes. Low-temperature electron paramagnetic resonance (EPR) spectra of low-spin ferric cytochromes c can be divided into two groups, depending on the spread of g values: the normal rhombic ones with small g anisotropy and g(max) below 3.2, and those featuring large g anisotropy with g(max) between 3.3 and 3.8, also denoted as highly axial low spin (HALS) species. Herein we present the detailed magnetic properties of cytochrome c(553) from Bacillus pasteurii (g(max) 3.36) and cytochrome c(552) from Nitrosomonas europaea (g(max) 3.34) over the pH range 6.2 to 8.2. Besides being structurally very similar, cytochrome c(553) shows the presence of a minor rhombic species at pH 6.2 (6 %), whereas cytochrome c(552) has about 25 % rhombic species over pH 7.5. The detailed Mössbauer analysis of cytochrome c(552) confirms the presence of these two low-spin ferric species (HALS and rhombic) together with an 8 % ferrous form with parameters comparable to the horse cytochrome c. Both EPR and Mössbauer data of axial cytochromes c with His-Met iron coordination are consistent with an electronic (d(xy))(2) (d(xz))(2) (d(yz))(1) ground state, which is typical for Type I model hemes.

Y25S Variant of Paracoccus pantotrophus Cytochrome cd1 Provides Insight into Anion Binding by d1 Heme and a Rare Example of a Critical Difference between Solution and Crystal Structures

Tyr25 is a ligand to the active site d1 heme in as isolated, oxidized cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. This form of the enzyme requires reductive activation, a process that involves not only displacement of Tyr25 from the d1 heme but also switching of the ligands at the c heme from bis-histidinyl to His/Met. A Y25S variant retains this bis-histidinyl coordination in the crystal of the oxidized state that has sulfate bound to the d1 heme iron. This Y25S form of the enzyme does not require reductive activation, an observation previously interpreted as meaning that the presence of the phenolate oxygen of Tyr25 is the critical determinant of the requirement for activation. This interpretation now needs re-evaluation because, unexpectedly, the oxidized as prepared Y25S protein, unlike the wild type, has different heme iron ligands in solution at room temperature, as judged by magnetic circular dichroism and electron spin resonance spectroscopies, than in the crystal. In addition, the binding of nitrite and cyanide to oxidized Y25S cytochrome cd1 is markedly different from the wild type enzyme, thus providing insight into the affinity of the oxidized d1 heme ring for anions in the absence of the steric barrier presented by Tyr25.

Temperature dependence of Q-band electron paramagnetic resonance spectra of nitrosyl heme proteins

The Q-band (35 GHz) electron paramagnetic resonance (EPR) spectra of nitrosyl hemoglobin (HbNO) and nitrosyl myoglobin (MbNO) were studied as a function of temperature between 19 K and 200 K. The spectra of both heme proteins show two classes of variations as a function of temperature. The first one has previously been associated with the existence of two paramagnetic species, one with rhombic and the other with axial symmetry. The second one manifests itself in changes in the g-factors and linewidths of each species. These changes are correlated with the conformational substates model and associate the variations of g-values with changes in the angle of the N(his)-Fe-N(NO) bond in the rhombic species and with changes in the distance between Fe and N of the proximal (F8) histidine in the axial species.

Characterization of cytochrome c 6 from the cyanobacterium Anabaena PCC 7119

A soluble monoheme c–type cytochrome c6 has been isolated from the cyanobacterium Anabaena PCC 7119. It is a basic protein, with a molecular mass of 9.7 kDa, which accepts electrons from Anabaena ferredoxin in the ferredoxin-NADP+reductase-dependent NADPH cytochrome c reductase activity assay. The turnover of the reaction has an optimum pH at 7.5. Flavodoxin can also replace ferredoxin in this assay, but with only 20% efficiency. Plastocyanin from Anabaena PCC 7119, as well as the c6 cytochromes from the green algae Chlorella fusca and Monoraphidium braunii are also shown to accept electrons from Anabaena ferredoxin. The reduction potential of cytochrome c6 at pH 6.7 was determined to be 338 mV and is pH dependent, with pKaox=8.4±0.1 and pKared≈9.5. The ferric and ferrous cytochrome forms and their pH equilibria have been studied using visible, EPR and 1H-NMR spectroscopies. The amino acid sequence and the visible and NMR spectroscopic data indicate that the heme iron has a methionine-histidine axial coordination in the pH range 5–11. However, the EPR data for the ferricytochrome are complex and show that in this pH range five distinct forms are present. Between pH 5 and 9 the spectrum is dominated by two rhombic species, with g–values at 2.94, 2.29, 1.43 and at 2.84, 2.34, 1.56, which interconvert with a pKa of 8.4. The NMR data also show a main interconversion between two cytochrome forms at this pH, which coincides with that determined from the pH dependence of the reduction potential. Both these forms were associated with a methionine-histidine heme-iron coordination by correlation with the visible and NMR spectral data, although having crystal field parameters atypical for this type of coordination. Anabaena cytochrome c6 is one more example of a heme protein for which the widely used crystal field analysis of the EPR data (truth diagram) fails to unequivocally determine the type of heme-iron ligation.

Identification of an Fe(III)‐dihydroxyphenylalanine Site in Recombinant Phosphomannose Isomerase from Candida albicans

Candida albicans phosphomannose isomerase (PMI) is a zinc metalloprotein of known crystal structure. When heterologously overexpressed in Escherichia coli, a blue protein that contains up to 0.5 iron atoms/PMI molecule could be isolated, with absorption maxima at 420 nm and 680 nm. These bands are reminiscent of ferric catecholate complexes, an assignment that has been confirmed by resonance Raman spectroscopy, and by reaction with Arnow's reagent, which is specific for the presence of 3,4-dihydroxyphenylalanine (Dopa). After enzymatic digestion of blue PMI, a peptide with the sequence DPHAXISG was isolated corresponding to residues Asp283-Gly290 in the amino acid sequence of C. albicans PMI, where the unidentified residue X287 is encoded by a tyrosine codon. It is proposed that iron and oxygen bring about hydroxylation of Tyr287 in PMI and that Fe(III) subsequently chelates the Dopa residue to give the characteristic absorption spectrum. The EPR spectrum of the blue protein suggests three iron environments in the protein, two in axial environments with E/D values approximately equal to 0.06 and 0.12 and one rhombic species. The nature of the iron co-ordination sites is discussed with the help of model systems and by comparison with other blue non-heme iron proteins.

Characterisation of cytochrome c 6 from Chlorella fusca

A c-type monohaem, cytochrome c6was isolated from a soluble extract of the green alga Chlorella fusca. The isolated protein shows an apparent molecular mass of 10 kDa by SDS-PAGE, but behaves as a dimer of 20.3 kDa in gel-filtration; the isoelectric point is 3.6. The N-terminal sequence shows high identity with other green algae cytochromes c6. The mid-point redox potential is about +350 mV between pH 5 and 9. The ferric and ferrous forms, and their pH equilibria, have been studied using visible, CD and EPR spectroscopies. The visible spectrum of the reduced cytochrome c6is typical of a c-type haem protein, with maxima at 274 nm, 318 nm (δ-peak), 416 nm (γ-peak), 522 nm (β-peak), 552–553 nm (α-peak). A 690 nm band, characteristic of a haem Met-His axial coordination of the haem group, is present in the oxidized form. At high pH values (≥ 8), cytochrome c6undergoes an alkaline transition, with a pKa of 8.7. Between pH 3 and 9 the EPR spectrum is dominated by two rhombic species, with g-values at 3.32, 2.05, 1.05 and 2.96, 2.30, 1.43, which interconvert with a pKaof 4. CD spectrum of Chlorella fusca cytochrome c6shows that the proteins must be mainly built up by α-helices. Even though there are similarities between Chlorella fusca cytochrome c6and that isolated from Monoraphidium braunii, no cross-reactivity with the antibodies raised against the Chlorella fusca cytochrome has been detected for the protein from Monoraphidium braunii.

ODMR and EPR investigations of Fe centers in KTaO3

The analysis of EPR spectra obtained from iron doped KTaO3 crystals in the as-grown state revealed three dominant iron centers: Fe3+-OI, axial Fe-centers with spinS = 3/2 and rhombic Fe3+. By comparison with data from literature possible assignments for the center withS = 3/2 are discussed. For the rhombic species the temperature dependence of the main parameters of the Spin- Hamiltonian was measured. The result makes it most plausible that only one rhombic iron center exists in KTaO3, in contrast with literature. The understanding of the EPR spectra allows us to assign transitions, observed at very low magnetic fields by optically detected magnetic resonance (ODMR), to this rhombic Fe center. On this basis, the magnetic circular dichroism (MCD) of this defect could be identified using the method of tagged-MCD. This spectrum is compared to the tagged-MCD of Fe3+-O1 and of axial Fe4+ centers, which may be generated metastably by optical charge transfer. Considerably different structures in the MCD spectra of both Fe3+ centers indicate different local surroundings and electronic states.

Access of ligands to the ferric center in lipoxygenase-1

A form of ferric lipoxygenase-1 has been isolated that gives an EPR spectrum that is dominated by a species of intermediate rhombicity (E/D = 0.065). This species is obtained in the presence of a number of buffers of high concentration and in the absence of fatty acid byproducts of the iron oxidation. The species is unstable over a period of one day with respect to symmetry of the iron. The EPR lineshapes of the unstable species are highly sensitive to the anionic composition of the buffer and to the addition of neutral ligands. These results suggest that newly formed ferric lipoxygenase has weak affinity for a number of ligands. Affinity of charged ligands for the iron center may provide a mechanism for charge compensation as the iron center alternates between ferric and ferrous in the catalytic cycle. We use spectral simulation to evaluate quantitatively the interaction of the ferric center with ligands and also show that a transition in the middle Kramers doublet makes a significant contribution to the EPR spectrum of the more rhombic species.

Intermediate species detected in oxidation reactions of FeTM(4)PyP 5+ with iodosylbenzene by EPR and UV-Vis spectroscopies

The iron porphyrin, tetrakis (4- N-methylpyridyl) porphyrinato iron(III) perchlorate (FeTM(4)PyP 5+) was studied as a catalyst for the oxidation reaction of an organic substrate by iodosylbenzene in organic solvents. The reaction system was studied by UV-Vis and EPR spectroscopic techniques at low temperature. The following intermediate species were detected: (i) iron(III)porphyrin rhombic species in the initial step of the reaction, (ii) oxo iron(IV)porphyrin (ferryl species) detected at 540 nm in the UV-Vis spectra and (iii) iron(III) N-oxides species (FeON) by EPR with g value 4.30. The species oxo iron(IV) porphyrin π-cation radical supposed to be present in the oxidation reaction was not detected in our experimental conditions. However, there is evidence for the existence of this species due to the reaction yields observed.

The cytochromes of anaerobically grown Escherichia coli. An electron-paramagnetic-resonance study of the cytochrome bd complex in situ

The e.p.r. signals attributable to a cytochrome bd-type ubiquinol:O2 oxidoreductase (cytochrome b-558-b-595-d) were studied in a cytoplasmic membrane preparation of Escherichia coli that had been grown on glycerol with fumarate as respiratory-chain oxidant. Two major high-spin ferric haem signals were resolved on the basis of their potentiometric behaviour: a rhombic high-spin species (gx = 6.25, gy = 5.54) was assigned to haem b-595, and an axial high-spin (gx = 5.97, gy = 5.96) species was assigned to the haem d. These signals titrated with Em.7 values of 154 and 261 mV respectively, corresponding closely to optically determined values for haem b-595 and haem d. At high potentials (greater than 300 mV) the rhombic species attributable to haem b-595 underwent a partial transition to a second rhombic species with g-values of 6.24 (gx) and 5.67 (gy). The high-spin ferric haem spectra were affected by O2, CO, cyanide and pH. A low-spin ferric haem signal was observed at g = 3.3 (gz), which titrated with an Em.7 of 226 mV, and this was assigned to haem b-558. The data support a model for cytochrome bd with two ligand-binding sites, a single haem d and a single haem b-595.

Paramagnetic metal species on highly dispersed rhodium/polyphosphine catalysts

ESR active paramagnetic species were used as a probe for the description of the metal centers in highly dispersed rhodium/polyphosphine catalysts. Magnetically dilute Rh(0) centers were observed at high P Rh s ] ( s = surface) ratios. Two different Rh(d0) diluted species were detected ( g 1 = 2.085, g 2 = 2.000; g′ 1 = 2.045, g′ 2 = 2.008, g′ 3 = 1.975) whose relative amounts vary with the P Rh s value. At lower P Rh s values, metal crystallites play a dominant role in giving broad unresolved signals. The metal particle size and the absolute Rh% are also related to the resonance shape. It is suggested that phosphine coordinates Rh centers, where a rhombic species corresponds to a higher number of coordinated phosphines. The results of the ESR investigation are in good agreement with previous studies on the same catalysts (G. Cocco, S. Enzo, F. Pina, and G. Strukul, J. Catal. 82, 160, 1983) performed by SAXS, WAXS, TGA, TPR, and infrared study of CO adsorption.

Electron paramagnetic resonance studies on Pseudomonas nitrosyl nitrite reductase. Evidence for multiple species in the electron paramagnetic resonance spectra of nitrosyl haemoproteins.

The e.p.r. spectra of reduced 14NO- and 15NO-bound Pseudomonas nitrite reductase have been investigated at pH 5.8 and 8.0 in four buffer systems. At pH 8.0, absorption spectra indicated that only the haem d1 was NO-bound, but, although quantification of the e.p.r. signals in all cases accounted for NO bound the the haem d1 in both subunits of the enzyme, the precise form of the signals varied with buffer and temperature. A rhombic species, with gx = 2.07, gz = 2.01 and gy = 1.96, represented in the low-temperature spectra seen in all the buffers was converted at high temperatures (approx. 200K) into a form showing a reduced anisotropy. Hyperfine splitting on the gz component of this rhombic signal indicated a nitrogen atom trans to NO and it is proposed that histidine provides the endogenous axial ligand for haem d1. At pH 5.8, absorption spectra indicated NO binding to both haems c and d1 and e.p.r. quantifications accounted for NO-bound haems c and d1 in both enzyme subunits. The e.p.r. spectra at pH 5.8 were generally similar to those at pH 8.0 with respect to g-values and hyperfine coupling constants, but were broader with less well defined hyperfine splittings. As at pH 8, rhombic signals present in spectra at low temperatures were converted to less anisotropic forms at high temperatures. The results are discussed in relation to work on model nitrosyl-protohaem complexes [Yoshimura, Ozaki, Shintani & Watanabe (1979) Arch. Biochem, Biophys. 193, 301-313]. No. e.p.r. signal was observed from oxidized NO-bound Pseudomonas nitrite reductase at pH 6.0, over the temperature range 6-100K.

EPR spectroscopy of soybean lipoxygenase-1 Determination of the zero-field splitting constants of high-spin Fe(III) signals from temperature and microwave frequency dependence

The zero-field splitting constants ( D) of the different components building up the high-spin Fe(III) EPR spectrum of lipoxygenase from soybeans were determined by two methods: (1) temperature dependence studies using the low-spin Fe(III) signal of cytochrome c at g 3 for accurate measuring of the temperature in the sample; (2) by establishing g- shift upon increasing the microwave frequency. The ranges of D for the axial and rhombic species contributing to the complex signal at g 6 are found to be 1.5–3.0 K and 1.8–4.4 K, respectively. The occurrence of such large ranges is attributed to variations in amount and number of species in the different samples. The combination of the applied methods offers a more generally applicable approach to the determination of zero-field splitting constants.