High-spin Signal Research Articles

High spin polarization and spin signal enhancement in non-local spin valves with Co–Fe alloy injectors and detectors

For applications such as spin accumulation sensors for next-generation hard disk drive read heads, and for fundamental research, it is desirable to increase the spin signal in metallic non-local spin valves, which are central devices in spintronics. To this end, here, we report on the integration of high-spin-polarization Co–Fe binary alloy ferromagnetic injectors and detectors in Al-based non-local spin valves. Room-temperature deposition on amorphous substrates from an alloy target is shown to generate smooth, polycrystalline (110-textured), solid-solution body-centered-cubic Co75Fe25 films, which we characterize by energy dispersive x-ray spectroscopy, x-ray diffraction, x-ray reflectivity, atomic force microscopy, and electronic transport. Simple integration into transparent-interface Al non-local spin valves is then shown to realize up to a factor of ∼5 enhancement of the spin signal relative to Co, with full quantitative analysis yielding strikingly temperature-independent current spin polarizations exceeding 60%. We make a detailed quantitative comparison of these values with prior literature, concluding that Co–Fe alloys present a remarkably facile route to higher spin polarization and spin signals in non-local spin valves, with minimal barrier to adoption.

Dissecting Electronic-Structural Transitions in the Nitrogenase MoFe Protein P-Cluster during Reduction.

The [8Fe-7S] P-cluster of nitrogenase MoFe protein mediates electron transfer from nitrogenase Fe protein during the catalytic production of ammonia. The P-cluster transitions between three oxidation states, PN, P+, P2+ of which PN↔P+ is critical to electron exchange in the nitrogenase complex during turnover. To dissect the steps in formation of P+ during electron transfer, photochemical reduction of MoFe protein at 231–263 K was used to trap formation of P+ intermediates for analysis by EPR. In complexes with CdS nanocrystals, illumination of MoFe protein led to reduction of the P-cluster P2+ that was coincident with formation of three distinct EPR signals: S = 1/2 axial and rhombic signals, and a high-spin S = 7/2 signal. Under dark annealing the axial and high-spin signal intensities declined, which coincided with an increase in the rhombic signal intensity. A fit of the time-dependent changes of the axial and high-spin signals to a reaction model demonstrates they are intermediates in the formation of the P-cluster P+ resting state and defines how spin-state transitions are coupled to changes in P-cluster oxidation state in MoFe protein during electron transfer.

Giant magnetoresistance in lateral metallic nanostructures for spintronic applications

In this letter, we discuss the shift observed in spintronics from the current-perpendicular-to-plane geometry towards lateral geometries, illustrating the new opportunities offered by this configuration. Using CoFe-based all-metallic LSVs, we show that giant magnetoresistance variations of more than 10% can be obtained, competitive with the current-perpendicular-to-plane giant magnetoresistance. We then focus on the interest of being able to tailor freely the geometries. On the one hand, by tailoring the non-magnetic parts, we show that it is possible to enhance the spin signal of giant magnetoresistance structures. On the other hand, we show that tailoring the geometry of lateral structures allows creating a multilevel memory with high spin signals, by controlling the coercivity and shape anisotropy of the magnetic parts. Furthermore, we study a new device in which the magnetization direction of a nanodisk can be detected. We thus show that the ability to control the magnetic properties can be used to take advantage of all the spin degrees of freedom, which are usually occulted in current-perpendicular-to-plane devices. This flexibility of lateral structures relatively to current-perpendicular-to-plane structures is thus found to offer a new playground for the development of spintronic applications.

Cross-linking of dicyclotyrosine by the cytochrome P450 enzyme CYP121 from Mycobacterium tuberculosis proceeds through a catalytic shunt pathway

CYP121, the cytochrome P450 enzyme in Mycobacterium tuberculosis that catalyzes a single intramolecular C-C cross-linking reaction in the biosynthesis of mycocyclosin, is crucial for the viability of this pathogen. This C-C coupling reaction represents an expansion of the activities carried out by P450 enzymes distinct from oxygen insertion. Although the traditional mechanism for P450 enzymes has been well studied, it is unclear whether CYP121 follows the general P450 mechanism or uses a different catalytic strategy for generating an iron-bound oxidant. To gain mechanistic insight into the CYP121-catalyzed reaction, we tested the peroxide shunt pathway by using rapid kinetic techniques to monitor the enzyme activity with its substrate dicyclotyrosine (cYY) and observed the formation of the cross-linked product mycocyclosin by LC-MS. In stopped-flow experiments, we observed that cYY binding to CYP121 proceeds in a two-step process, and EPR spectroscopy indicates that the binding induces active site reorganization and uniformity. Using rapid freeze-quenching EPR, we observed the formation of a high-spin intermediate upon the addition of peracetic acid to the enzyme-substrate complex. This intermediate exhibits a high-spin (S = 5/2) signal with g values of 2.00, 5.77, and 6.87. Likewise, iodosylbenzene could also produce mycocyclosin, implicating compound I as the initial oxidizing species. Moreover, we also demonstrated that CYP121 performs a standard peroxidase type of reaction by observing substrate-based radicals. On the basis of these results, we propose plausible free radical-based mechanisms for the C-C bond coupling reaction.

Nanosecond spin relaxation times in single layer graphene spin valves with hexagonal boron nitride tunnel barriers

We present an experimental study of spin transport in single layer graphene using atomic sheets of hexagonal boron nitride (h-BN) as a tunnel barrier for spin injection. While h-BN is expected to be favorable for spin injection, previous experimental studies have been unable to achieve spin relaxation times in the nanosecond regime, suggesting potential problems originating from the contacts. Here, we investigate spin relaxation in graphene spin valves with h-BN barriers and observe room temperature spin lifetimes in excess of a nanosecond, which provides experimental confirmation that h-BN is indeed a good barrier material for spin injection into graphene. By carrying out measurements with different thicknesses of h-BN, we show that few layer h-BN is a better choice than monolayer for achieving high non-local spin signals and longer spin relaxation times in graphene.

Using domain walls to perform non-local measurements with high spin signal amplitudes

Standard non-local measurements require lateral spin-valves with two different ferromagnetic electrodes, to create and to detect the spin accumulation. Here we show that non-local measurements can also be performed in a cross-shaped nanostructure, made of a single ferromagnetic wire connected to an orthogonal non-magnetic wire. A magnetic domain wall located underneath the ferromagnetic/non-magnetic interface is used to control the magnetizations of the injection and detection zones. As these zones can be very close, our results display spin signals possessing amplitudes larger than those obtained in conventional non-local measurements. We also show that this method can be used as a domain wall detection technique.

Effect of Sequestering Intrinsic Iron on the Electron Paramagnetic Resonance Signals in Powdered Soy Proteins

This investigation examined iron in powdered soy protein products using electron paramagnetic resonance (EPR) spectroscopy, and the effect that selectively binding free iron in isolated soy protein (ISP) had on the occurrence of metastable radicals in powdered soy proteins. EPR analyses of soybean defatted flour, commercial ISP and laboratory ISP samples revealed a peak at g = 4.3 characteristic of high-spin ferric iron in a rhombic-coordinated environment. Commercial ISP samples examined contained higher levels of the rhombic ferric iron than laboratory-prepared ISP samples. During the first 6 wk of storage the primary singlet EPR signal at g = 2.0049 in the commercial ISP samples approximately doubled, and the laboratory prepared samples increased by about 9-fold. The EPR signal was initially about 4-times higher in the freshly prepared commercial samples compared to the corresponding laboratory ISP. Laboratory ISP samples prepared with added deferoxamine to sequester endogenous iron exhibited a large increase in the high-spin ferric iron EPR signal at g = 4.3. ISP treated with deferoxamine also exhibited a multiple-line EPR signal at about g = 2.007, instead of the typical singlet signal at g = 2.0049. The power at which the signal amplitude was half-saturated also changed from about 1 mW in the control ISP to about 20 mW in the deferoxamine treated ISP. The multiple-line EPR spectrum from the ISP treated with deferoxamine increased during storage over a 6-wk period by about 6-fold. The observed changes in EPR line-shape, g-value, and power saturation with the deferoxamine treatment indicate that the primary free-radical signal in powdered ISP samples may be from stabilized tyrosine radicals with spin densities distributed over the aromatic ring.

Role of the Iron Axial Ligands of Heme Carrier HasA in Heme Uptake and Release

The hemophore protein HasA from Serratia marcescens cycles between two states as follows: the heme-bound holoprotein, which functions as a carrier of the metal cofactor toward the membrane receptor HasR, and the heme-free apoprotein fishing for new porphyrin to be taken up after the heme has been delivered to HasR. Holo- and apo-forms differ for the conformation of the two loops L1 and L2, which provide the axial ligands of the iron through His(32) and Tyr(75), respectively. In the apo-form, loop L1 protrudes toward the solvent far away from loop L2; in the holoprotein, closing of the loops on the heme occurs upon establishment of the two axial coordination bonds. We have established that the two variants obtained via single point mutations of either axial ligand (namely H32A and Y75A) are both in the closed conformation. The presence of the heme and one out of two axial ligands is sufficient to establish a link between L1 and L2, thanks to the presence of coordinating solvent molecules. The latter are stabilized in the iron coordination environment by H-bond interactions with surrounding protein residues. The presence of such a water molecule in both variants is revealed here through a set of different spectroscopic techniques. Previous studies had shown that heme release and uptake processes occur via intermediate states characterized by a Tyr(75)-iron-bound form with open conformation of loop L1. Here, we demonstrate that these states do not naturally occur in the free protein but can only be driven by the interaction with the partner proteins.

Functional Characterization of the FoxE Iron Oxidoreductase from the Photoferrotroph Rhodobacter ferrooxidans SW2

Photoferrotrophy is presumed to be an ancient type of photosynthetic metabolism in which bacteria use the reducing power of ferrous iron to drive carbon fixation. In this work the putative iron oxidoreductase of the photoferrotroph Rhodobacter ferrooxidans SW2 was cloned, purified, and characterized for the first time. This protein, FoxE, was characterized using spectroscopic, thermodynamic, and kinetic techniques. It is a c-type cytochrome that forms a trimer or tetramer in solution; the two hemes of each monomer are hexacoordinated by histidine and methionine. The hemes have positive reduction potentials that allow downhill electron transfer from many geochemically relevant ferrous iron forms to the photosynthetic reaction center. The reduction potentials of the hemes are different and are cross-assigned to fast and slow kinetic phases of ferrous iron oxidation in vitro. Lower reactivity was observed at high pH and may contribute to prevent ferric iron precipitation inside or at the surface of the cell. These results help fill in the molecular details of a metabolic process that likely contributed to the deposition of precambrian banded iron formations, globally important sedimentary rocks that are found on every continent today.

Characterization of dye-decolorizing peroxidases from Rhodococcus jostii RHA1.

The soil bacterium Rhodococcus jostii RHA1 contains two dye-decolorizing peroxidases (DyPs) named according to the subfamily they represent: DypA, predicted to be periplasmic, and DypB, implicated in lignin degradation. Steady-state kinetic studies of these enzymes revealed that they have much lower peroxidase activities than C- and D-type DyPs. Nevertheless, DypA showed 6-fold greater apparent specificity for the anthraquinone dye Reactive Blue 4 (k(cat)/K(m) = 12800 ± 600 M(-1) s(-1)) than either ABTS or pyrogallol, consistent with previously characterized DyPs. By contrast, DypB showed the greatest apparent specificity for ABTS (k(cat)/K(m) = 2000 ± 100 M(-1) s(-1)) and also oxidized Mn(II) (k(cat)/K(m) = 25.1 ± 0.1 M(-1) s(-1)). Further differences were detected using electron paramagnetic resonance (EPR) spectroscopy: while both DyPs contained high-spin (S = (5)/(2)) Fe(III) in the resting state, DypA had a rhombic high-spin signal (g(y) = 6.32, g(x) = 5.45, and g(z) = 1.97) while DypB had a predominantly axial signal (g(y) = 6.09, g(x) = 5.45, and g(z) = 1.99). Moreover, DypA reacted with H(2)O(2) to generate an intermediate with features of compound II (Fe(IV)═O). By contrast, DypB reacted with H(2)O(2) with a second-order rate constant of (1.79 ± 0.06) × 10(5) M(-1) s(-1) to generate a relatively stable green-colored intermediate (t(1/2) ∼ 9 min). While the electron absorption spectrum of this intermediate was similar to that of compound I of plant-type peroxidases, its EPR spectrum was more consistent with a poorly coupled protein-based radical than with an [Fe(IV)═O Por(•)](+) species. The X-ray crystal structure of DypB, determined to 1.4 Å resolution, revealed a hexacoordinated heme iron with histidine and a solvent species occupying axial positions. A solvent channel potentially provides access to the distal face of the heme for H(2)O(2). A shallow pocket exposes heme propionates to the solvent and contains a cluster of acidic residues that potentially bind Mn(II). Insight into the structure and function of DypB facilitates its engineering for the improved degradation of lignocellulose.

Effect of texture alteration by thin film fabrication on the spin crossover of [Fe(3-Br-phen)2(NCS)2]·0.5CH3OH

A mononuclear complex of composition [Fe(3-Br-phen)2(NCS)2]·0.5CH3OH which exhibits a gradual, incomplete spin crossover (SCO) in bulk sample (1) was selected to study the effect of alteration of particle size and shape on SCO properties. Thin films (2) were fabricated using spin coating technique on quartz substrates and silicon wafers. Morphology analysis by scanning electron micrographs shows a drastic reduction in particles size and deformation in shape compared to the bulk sample. Nearly spherical particles of 200-500 nm diameter were obtained and further reduction in size down to 50-120 nm were obtained with increase in spin coating speed. A thin film on a quartz substrate was studied by 57Fe Mössbauer spectroscopy in transmission mode which shows only a low-spin signal as opposed to a dominant high-spin signal in bulk sample thus hinting out the effect of texture modification on increasing the ligand field strength.

The spin-transition properties of Fe(III) complexes with hetarylformazan in an ion-exchange polymer: An EPR study

For the first time, the EPR method was used to reveal and study the special features of spin-transition processes between high-spin and low-spin iron(III) complexes with hetarylformazans immobilized on an ion-exchange polymer. An analysis of completely reversible temperature dependences of EPR line positions, widths, and integral intensities in the spectra of high-spin and low-spin complexes allowed four temperature intervals to be identified. These intervals corresponded to preparative periods of spin-transition processes (450–275 K, intervals I and II), their appearance (275–230 K, interval III), and occurrence (230–100 K, interval IV). Local concentrations and spin exchange frequencies in clusters were estimated. Effects related to high-spin complex EPR signal shifts during temperature changes and to the duration of sample storage were revealed. High-spin complexes were found to be very sensitive to external actions, as distinct from very stable low-spin complexes. Experimental EPR observations obtained for ion-exchange polymer satisfied the concept of the nucleation and growth of domains in the spin-transition process.

Biochemical and Spectroscopic Properties of Cyanide-Insensitive Quinol Oxidase from Gluconobacter oxydans

Cyanide-insensitive quinol oxidase (CioAB), a relative of cytochrome bd, has no spectroscopic features of hemes b(595) and d in the wild-type bacteria and is difficult to purify for detailed characterization. Here we studied enzymatic and spectroscopic properties of CioAB from the acetic acid bacterium Gluconobacter oxydans. Gluconobacter oxydans CioAB showed the K(m) value for ubiquinol-1 comparable to that of Escherichia coli cytochrome bd but it was more resistant to KCN and quinone-analogue inhibitors except piericidin A and LL-Z1272gamma. We obtained the spectroscopic evidence for the presence of hemes b(595) and d. Heme b(595) showed the alpha peak at 587 nm in the reduced state and a rhombic high-spin signal at g = 6.3 and 5.5 in the air-oxidized state. Heme d showed the alpha peak at 626 and 644 nm in the reduced and air-oxidized state, respectively, and an axial high-spin signal at g = 6.0 and low-spin signals at g = 2.63, 2.37 and 2.32. We found also a broad low-spin signal at g = 3.2, attributable to heme b(558). Further, we identified the presence of heme D by mass spectrometry. In conclusion, CioAB binds all three ham species present in cytochrome bd quinol oxidase.

Bis-methionine Ligation to Heme Iron in the Streptococcal Cell Surface Protein Shp Facilitates Rapid Hemin Transfer to HtsA of the HtsABC Transporter

The surface protein Shp of Streptococcus pyogenes rapidly transfers its hemin to HtsA, the lipoprotein component of the HtsABC transporter, in a concerted two-step process with one kinetic phase. The structural basis and molecular mechanism of this hemin transfer have been explored by mutagenesis and truncation of Shp. The heme-binding domain of Shp is in the amino-terminal region and is functionally active by itself, although inclusion of the COOH-terminal domain speeds up the process approximately 10-fold. Single alanine replacements of the axial methionine 66 and 153 ligands (Shp(M66A) and Shp(M153A)) cause formation of pentacoordinate hemin-Met complexes. The association equilibrium constants for hemin binding to wild-type, M66A, and M153A Shp are 5,300, 22,000, and 38 microM(-1), respectively, showing that the Met(153)-Fe bond is critical for high affinity binding and that Met(66) destabilizes hemin binding to facilitate its rapid transfer. Shp(M66A) and Shp(M153A) rapidly bind to hemin-free HtsA (apoHtsA), forming stable transfer intermediates. These intermediates appear to be Shp-hemin-HtsA complexes with one axial ligand from each protein and decay to the products with rate constants of 0.4-3 s(-1). Thus, the M66A and M153A replacements alter the kinetic mechanism and unexpectedly slow down hemin transfer by stabilizing the intermediates. These results, in combination with the structure of the Shp heme-binding domain, allow us to propose a "plug-in" mechanism in which side chains from apoHtsA are inserted into the axial positions of hemin in Shp to extract it from the surface protein and pull it into the transporter active site.

The Mechanism of Direct Heme Transfer from the Streptococcal Cell Surface Protein Shp to HtsA of the HtsABC Transporter

The heme-binding proteins Shp and HtsA are part of the heme acquisition machinery found in Streptococcus pyogenes. The hexacoordinate heme (Fe(II)-protoporphyrin IX) or hemochrome form of holoShp (hemoShp) is stable in air in Tris-HCl buffer, pH 8.0, binds to apoHtsA with a K(d) of 120 +/- 18 microm, and transfers its heme to apoHtsA with a rate constant of 28 +/- 6s(-1) at 25 degrees C, pH 8.0. The hemoHtsA product then autoxidizes to the hexacoordinate hemin (Fe(III)-protoporphyrin IX) or hemichrome form (hemiHtsA) with an apparent rate constant of 0.017 +/- 0.002 s(-1). HemiShp also rapidly transfers hemin to apoHtsA through a hemiShp.apoHtsA complex (K(d) = 48 +/- 7 microM) at a rate approximately 40,000 times greater than the rate of simple hemin dissociation from hemiShp into solvent (k(transfer) = 43 +/- 3s(-1) versus k(-hemin) = 0.0003 +/- 0.00006 s(-1)). The rate constants for hemin binding to and dissociation from HtsA (k'(hemin) approximately 80 microm(-1) s(-1), k(-hemin) = 0.0026 +/- 0.0002 s(-1)) are 50- and 10-fold greater than the corresponding rate constants for Shp (k(hemin) approximately 1.6 microM(-1) s(-1), k(-hemin) = 0.0003 s(-1)), which implies that HtsA has a more accessible active site. However, the affinity of apoHtsA for hemin (k(hemin) approximately 31,000 microm(-1)) is roughly 5-fold greater than that of apoShp (k(hemin) approximately 5,300 microM(-1)), accounting for the net transfer from Shp to HstA. These results support a direct, rapid, and affinity-driven mechanism of heme and hemin transfer from the cell surface receptor Shp to the ATP-binding cassette transporter system.

Chemically distinct Ni sites in the A-cluster in subunit beta of the acetyl-CoA decarbonylase/synthase complex from Methanosarcina thermophila: Ni L-edge absorption and X-ray magnetic circular dichroism analyses.

The 5-subunit-containing acetyl-CoA decarbonylase/synthase (ACDS) complex plays an important role in methanogenic Archaea that convert acetate to methane, by catalyzing the central reaction of acetate C-C bond cleavage in which acetyl-CoA serves as the acetyl donor substrate reacting at the ACDS beta subunit active site. The properties of Ni in the active site A-cluster in the ACDS beta subunit from Methanosarcina thermophila were investigated. A recombinant, C-terminally truncated form of the beta subunit was employed, which mimics the native subunit previously isolated from the ACDS complex, and contains an A-cluster composed of an [Fe(4)S(4)] center bridged to a binuclear Ni-Ni site. The electronic structures of these two Ni were studied using L-edge absorption and X-ray magnetic circular dichroism (XMCD) spectroscopy. The L-edge absorption data provided evidence for two distinct Ni species in the as-isolated enzyme, one with low-spin Ni(II) and the other with high-spin Ni(II). XMCD spectroscopy confirmed that the species producing the high-spin signal was paramagnetic. Upon treatment with Ti(3+) citrate, an additional Ni species emerged, which was assigned to Ni(I). By contrast, CO treatment of the reduced enzyme converted nearly all of the Ni in the sample to low-spin Ni(II). The results implicate reaction of a high-spin tetrahedral Ni site with CO to form an enzyme-CO adduct transformed to a low-spin Ni(II) state. These findings are discussed in relation to the mechanism of C-C bond activation, in connection with the model of the beta subunit A-cluster developed from companion Ni and Fe K edge, XANES, and EXAFS studies.

EPR Spectrometry of Cytochrome P450 2B4: Effects of Mutations and Substrate Binding

The EPR spectra of NH2-terminal-truncated P450 cytochrome 2B4 and of several active site mutants that were previously shown to be profoundly altered in catalytic properties were determined. From these spectra it was seen that the truncated P450 2B4, like the full length cytochrome, exists as the low spin ferric form, but upon mutation of threonine 302 to alanine approximately 40% of the cytochrome is present as the high spin ferric form (g ≈ 8, 4, 2). A similar situation was observed in the double mutant E310L T302A, but not in the single mutant E301L. A rhombic high spin signal (g ≈ 8, 4, 2) was observed when a substrate such as styrene, benzphetamine, or cyclohexane was added to the truncated cytochrome. Accompanying this change was the appearance of a signal at g = 1.98. Conversely, an axial high spin signal was observed (g ≈ 6, 6, 2) when cyclohexanecarboxaldehyde or 3-phenylpropionaldehyde was added to the truncated P450 2B4.

Interaction between 6-hydroxydopamine and transferrin: "Let my iron go".

The dopamine analogue 6-hydroxydopamine (6-OHDA) is selectively toxic to catecholaminergic neurons. Because of its selectivity for neuroblastic cells in the sympathetic nervous system lineage, 6-OHDA has been suggested as a chemotherapeutic agent for targeted treatment of patients with neuroblastoma. We tested the hypothesis that the toxicity of 6-OHDA is caused by its interaction with serum ferric transferrin (Fe-TF) resulting in release of iron. We further hypothesized that this iron, through its redox-cycling by 6-OHDA, triggers generation of reactive oxygen species. 6-OHDA-induced release of iron from Fe-TF was demonstrated by: (1) low-temperature EPR spectroscopic evidence for decay of the characteristic Fe-TF signal (g = 4.3) and appearance of the high-spin signal from iron chelated by 6-OHDA oxidation products; (2) spectrophotometric detection of complexing of iron with the Fe(2+) chelator ferrozine; (3) redox-cycling of ascorbate yielding EPR-detectable ascorbate radicals; and (4) generation of hydroxyl radicals as evidenced by EPR spectroscopy of their adduct with a spin trap, 5, 5'-dimethylpyrroline oxide (DMPO) (DMPO-OH). Our low-temperature EPR studies showed that in human plasma, 6-OHDA caused iron release only under nitrogen gas but not under air or oxygen. The absence of a 6-OHDA effect in plasma under aerobic conditions was most likely due to its ferroxidase activity [with consequent reuptake of Fe(III) by apoTF] and catalytic oxidation of 6-OHDA by ceruloplasmin. Modeling of these plasma activities by a stable nitroxide radical, 2,2,6, 6-tetramethyl-1-piperidinyloxy (TEMPOL), resulted in protection of plasma Fe-TF against iron release under nitrogen. Parenteral administration of 6-OHDA to mice resulted in iron release from Fe-TF as evidenced by transformation of the Fe-TF low-temperature EPR signal that was indistinguishable from that seen in in vitro models. In addition, administration of the iron chelator deferoxamine (DFO) to mice prior to administration of toxic doses of 6-OHDA resulted in a decrease in activity impairment of mice as compared to that seen with 6-OHDA alone. These findings underscore the physiological and pharmacological relevance of 6-OHDA-mediated iron release from Fe-TF and suggest that iron chelators (DFO) may be used for prevention of 6-OHDA toxicity.

A New Method for Quantitation of Spin Concentration by EPR Spectroscopy: Application to Methemoglobin and Metmyoglobin

A new method of EPR spectral analysis is developed to quantitate overlapping signals. The method requires double integration of a number of spectra containing the signals in different proportions and the subsequent solution of a system of linear equations. The result gives the double integral values of the individual lines, which can then be further used to find the concentrations of all the paramagnetic species present. There is no requirement to deconvolute the whole spectrum into its individual components. The method is employed to quantify different heme species in methemoglobin and metmyoglobin preparations. A significantly greater intensity of the high-spin signal in metmyoglobin, compared to methemoglobin at the same heme concentration, is shown to be due to larger amounts of low-spin forms in methemoglobin. Three low-spin types in methemoglobin and two in metmyoglobin are present in these samples. When their calculated concentrations are added to those of the high-spin forms, the results correspond to the total heme concentrations obtained by optical spectroscopy.

Azide- and cyanide-binding to the Escherichia coli bd-type ubiquinol oxidase studied by visible absorption, EPR and FTIR spectroscopies.

Cytochrome bd-type ubiquinol oxidase contains two hemes b (b(558) and b(595)) and one heme d as the redox metal centers. To clarify the structure of the reaction center, we analyzed Escherichia coli cytochrome bd by visible absorption, EPR and FTIR spectroscopies using azide and cyanide as monitoring probes for the exogenous ligand binding site. Azide-binding caused the appearance of a new EPR low-spin signal characteristic of ferric iron-chlorin-azide species and a new visible absorption band at 647 nm. However, the bound azide ((14)N(3)) anti-symmetric stretching infrared band (2, 010.5 cm(-1)) showed anomalies upon (15)N-substitutions, indicating interactions with surrounding protein residues or heme b(595) in close proximity. The spectral changes upon cyanide-binding in the visible region were typical of those observed for ferric iron-chlorin species with diol substituents in macrocycles. However, we found no indication of a low-spin EPR signal corresponding to the ferric iron-chlorin-cyanide complexes. Instead, derivative-shaped signals at g = 3.19 and g = 7.15, which could arise from the heme d(Fe(3+))-CN-heme b(595)(Fe(3+)) moiety, were observed. Further, after the addition of cyanide, a part of ferric heme d showed the rhombic high-spin signal that coexisted with the g(z) = 2.85 signal ascribed to the minor heme b(595)-CN species. This indicates strong steric hindrance of cyanide-binding to ferric heme d with the bound cyanide at ferric heme b(595).