Low-temperature Electron Paramagnetic Resonance Research Articles

Photochemistry of the IrCl 2−6 complex in methanol matrices

Low-temperature spectrophotometry and electron spin resonance (ESR) were used to study the IrC 2− 6 photochemistry in methanol matrices frozen at 77 K. Photoreaction gives rise to the absorption band of an IrCl 3− 6 complex with a maximum at 215 nm and to a new band with a maximum at 287 nm that is unobservable during IrCl 2− 6 photolysis in solutions at room temperature. The appearance of this new band is assigned to the formation of a weak complex between the .CH 2OH radical and IrCl 3− 6 ion (i.e. a IrCl 3− 6 … .CH 2OH radical complex). The irradiation of methanol matrix with IrCl 2− 6 gives rise to the .CH 2OH radical signal in the ESR spectrum with additional splitting which can be attributed to the partial delocalization of spin density from radical to the IrCl 3− 6 complex and the appearance of hyperfine splitting (HFS) on the iridium nucleus. When the matrix is heated to 115 K the IrCl 3− 6 … .CH 2OH complex disappears.

Two modes of ligand binding in maltose-binding protein of Escherichia coli. Electron paramagnetic resonance study of ligand-induced global conformational changes by site-directed spin labeling.

Binding of ligands to the maltose-binding protein (MBP) of Escherichia coli often causes a global conformational change involving the closure of its two lobes. We have introduced a cysteine residue onto each of these lobes by site-directed mutagenesis and modified these residues with spin labels. Using EPR spectroscopy, we examined the changes, caused by the ligand binding, in distance between the two spin labels, hence between the two lobes. The binding of both maltose and maltotetraose induced a considerable closure of the N- and C-terminal lobes of MBP. Little closure occurred upon the binding of maltotetraitol or beta-cyclodextrin. Previous study by fluorescence and UV differential absorbance spectroscopy (Hall, J. A., Gehring, K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17605-17609) showed that maltose and a large portion of maltotetraose bound to MBP via one mode (R mode or "end-on" mode), which is physiologically active and leads to the subsequent transport of the ligands across the cytoplasmic membrane. In contrast, maltotetraitol and beta-cyclodextrin bound to MBP via a different mode (B mode or "middle" mode), which is physiologically inactive. The present work suggests that the B mode is nonproductive because ligands binding in this manner prevent the closure of the two domains of MBP, and, as a result, the resulting ligand-MBP complex is incapable of interacting properly with the inner membrane-associated transporter complex.

EPR Studies of Photoreduction of Ni/TiO2 Catalysts

AbstractIrradiations of Ni/TiO2 catalyst by UV in hydrogen at 77 K produced not only Ni+ ions on the catalyst surface, but also Ni3+ and Ti3+ species in bulk or near the interface between nickel and titania. These photo‐generated species were detected and characterized by low temperature electron paramagnetic resonance (EPR) spectroscopy. Relative spin concentrations of the photogenerated paramagnetic species (Nin+ and Ti3+) varied with the nickel content in titania. A high nickel content in the sample resulted in a high peak intensity ratio of Nin+ to Ti3+. It was found that the photoinduced self‐redox reaction of Ni2+ ions to form Ni+ and Ni3+ ions has a priority over the photoreduction of Ti4+ to Ti3+ ions. The characteristic EPR spectrum of the Ni3+ (3d7) ions with g1 = 2.268, g2 = 2.237, and g3 = 2.045 indicates that the Ni3+ ions are most likely located in the substitutional sites of TiO2, possibly near the surface rutile phase. The Ni+ species (3d9) with g4 = 2.130 and g1 = 2.063 are on the surface of TiO2. Both Ni+ and Ni3+ ions are quite stable in hydrogen. The Ni3+ ions seem to be responsible for anchoring the nickel ions onto titania and stablizing the Ni+ species on the surface. The Ni+ ions are thus free from oxygen poisoning and still show a high activity toward olefin oligomerization.

In vivoDetection of Nitric Oxide and NOxSpecies Usingex vivoElectron Paramagnetic Resonance Spectroscopy

Nitric oxide is a paramagnetic molecule that exhibits high affinity for heme proteins, forming a mixture of nitrosylated valency hybrids that can be precisely characterized using low-temperature electron paramagnetic resonance spectroscopy. In red blood cells, nitric oxide will form paramagnetic complexes with deoxyhemoglobin or be oxidized to nitrite and nitrate by oxyhemoglobin with formation of paramagnetic methemoglobin. We have developed a technique to monitor temporal changes in local nitric oxide productionin vivo,by sampling arterial and venous blood across a tissue bed and measuring changes in hemoglobin–nitric oxide and methemoglobin concentrationex vivousing electron paramagnetic resonance spectroscopy. Changes in local nitric oxide concentration are further clarified by quantifying plasma nitrite/nitrate levels. With these techniques, we have observed that whole body hyperthermia increases portal venous concentrations of hemoglobin–nitric oxide and organic radicals that are consistent with enhanced nitric oxide synthase activity, cytotoxic leukocyte activity, and cellular oxidative stress in splanchnic tissues.

The Effects of Lattice Water on Free Radical Yields in X-Irradiated Crystalline Pyrimidines and Purines: A Low-Temperature Electron Paramagnetic Resonance Investigation

The hydration layer of DNA increases the target size of DNA with respect to the formation of direct-type damage by ionizing radiation. The mechanisms that give rise to this increase are being investigated by EPR spectroscopy. To determine these mechanisms, it is necessary to distinguish between the change in sample mass and changes in packing/conformation brought about by the change in the level of hydration. Certain model compounds that crystallize as hydrates provide a system where the effects of mass and packing can be discerned. Three such hydrate crystals were used in this work: barbituric acid dihydrate (BA:2H2O), inosine dihydrate (IR:2H2O) and thymine monohydrate (T:H2O). The free radical yields (+/-25%) in the native crystals at 7-11 K are 0.08, 0.03 and 0.02, respectively. Removal of the lattice water leaves behind an ordered lattice and results in free radical yields of 0.08, 0.03 and < 0.004, respectively. Thus removal of the lattice water does not affect the free radical yield in BA:2H2O or IR:2H2O but decreases the free radical yield in T:H2O by an order of magnitude. Based on these observations and the known crystal packing, we conclude that the hydrogen bonding network is a major factor in determining the distribution and yield of trapped free radicals. We ascribe this to the importance of proton transfer processes which act to reduce the probability of radical combination. Consistent with this conclusion are the types of free radicals trapped in these crystalline materials before and after dehydration. From these results, we argue that a major determinant of free radical yields in solidstate samples of DNA constituents is molecular packing. In addition, the absence of HO. radicals trapped in single crystals of BA:2H2O provides an upper limit for the yield of trapped HO. of less than 10(-4) mumol/J. This supports the thesis that at < 77 K direct ionization of those waters associated directly with a pyrimidine or purine results in hole transfer to that molecule. Hydroxyl radical formation on a water adjacent to a DNA base is predicted to be negligible.

Role of calcium in maintaining the heme environment of manganese peroxidase.

We previously demonstrated that manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium was very susceptible to thermal inactivation due to the loss of calcium from the enzyme [Sutherland & Aust (1996) Arch. Biochem. Biophys. 332, 128-134]. The structural changes that occur during thermal inactivation and the release of calcium from manganese peroxidase have now been characterized. Thermal inactivation caused distinct alterations in the heme environment and slight changes in the overall protein structure, both of which were reversed upon reactivation of the enzyme with calcium. The absorption spectrum of inactivated manganese peroxidase was similar to that of low-spin ferric heme proteins, indicating that a sixth ligand had bound to the distal side of the heme iron. Consistent with disruption of the distal heme environment, thermally inactivated manganese peroxidase did not react with hydrogen peroxide to form compound I. The inactive enzyme exhibited a pH-dependent absorption transition with a pKa of 5.7. Studies involving imidazole indicated that the sixth ligand may be a distal histidine. Low-temperature electron paramagnetic resonance spectroscopy confirmed that the heme iron of the inactivated form of manganese peroxidase was predominantly in a low-spin state. The near-ultraviolet/visible circular dichroism spectrum also supported the proposed formation of a highly symmetric bis(imidazole) heme complex upon thermal inactivation of the enzyme. A recombinant manganese peroxidase, in which the distal calcium binding site was altered such that calcium binding would be minimized, was also characterized. This enzyme, D47A, had the same catalytic and spectroscopic properties and calcium content as thermally inactivated manganese peroxidase. Therefore, the inactivation and structural changes that occurred during thermal incubation of manganese peroxidase could be explained by the loss of the distal calcium.

Kinetics and motional dynamics of spin-labeled yeast iso-1-cytochrome c: 1. Stopped-flow electron paramagnetic resonance as a probe for protein folding/unfolding of the C-terminal helix spin-labeled at cysteine 102.

The kinetics of chemically induced folding and unfolding processes in spin-labeled yeast iso-1-cytochrome c were measured by stopped-flow electron paramagnetic resonance (EPR). Stopped-flow EPR, based on a new dielectric resonator structure [Sienkiewicz, A., Qu, K., & Scholes, C. P. (1994) Rev. Sci. Instrum. 65, 68-74], gives a new temporal component to probing nanosecond molecular tumbling motions that are modulated by macromolecular processes requiring time resolution of milliseconds to seconds. The stopped-flow EPR technique presented in this work is a kinetic technique that has not been previously used with such a time resolution on spin-labeled systems, and it has the potential for application to numerous spin-labeled sites in this and other proteins. The cysteine-specific spin-label, methanethiosulfonate spin-label (MTSSL), was attached to yeast iso-1-cytochrome c at the single naturally occurring cysteine102, and the emphasis for this work was on this disulfide-attached spin-labeled prototype. This probe has the advantage of reflecting the protein tertiary fold, as shown by recent, systematic site-directed spin labeling of T4 lysozyme [Mchaourab, H. S. Lietzow, M. A., Hideg, K., & Hubbell, W. L. (1996) Biochemistry 35, 7692-7704], and protein backbone dynamics, as also shown by model peptide studies [Todd, A. P., & Millhauser, G. L. (1991) Biochemistry 30, 5515-5523]. The C-terminal cytochrome c helix where the label is attached is thought to be critical in the initial steps of protein folding and unfolding. Stopped-flow EPR resolved the monoexponential, guanidinium-induced unfolding process at pH 6.5 with an approximately 20 ms time constant; this experiment required less than 150 microL of 80 microM spin-labeled protein. We observed an approximately 50-fold decrease of this unfolding time from the 1 s range to the 20 ms time range as the guanidinium denaturant concentration was increased from 0.6 to 2.0 M. The more complex refolding kinetics of our labeled cytochrome were studied by stopped-flow EPR at pH 5.0 and 6.5. The spin probe showed a fast kinetic process compatible with the time range over which hydrogen/deuterium amide protection indicates helix formation; this process was monoexponential at pH 5.0. At pH 6.5, there was evidence of an additional slower kinetic phase resolved by stopped-flow EPR and by heme-ligation-sensitive UV-Vis that indicated a slower folding where heme misligation may be involved. Since the disulfide-attached probe has reported folding and backbone dynamics in other systems, the implication is that our kinetic experiments were directly sensing events of the C-terminal helix formation and possibly the N- and C-terminal helical interaction. The cysteine-labeled protein was also studied under equilibrium conditions to characterize probe mobility and the effect of the probe on protein thermodynamics. The difference in spin probe mobility between folded and denatured protein was marked, and in the folded protein, the motion of the probe was anisotropically restricted. The motion of the attached nitroxide in the folded protein appears to be restricted about the carbon and sulfur bonds which tether it to the cysteine. The original point of cysteine sulfur attachment is approximately 11 A from the heme iron within the C-terminal helix near its interface with the N-terminal helix, but the low-temperature EPR spin probe line width showed that the probe lies more distant (> 15 A) from the heme iron. By all physical evidence, the protein labeled at cysteine102 folded, but the spin probe in this prototype system perturbed packing which lowered the thermal melting temperature, the free energy of folding, the guanidinium concentration at the midpoint of the unfolding transition, the m parameter of the denaturant, and the helical CD signature. This study prepares the way for study of protein folding/unfolding kinetics using EPR spectroscopy of spin-labels placed at specific cysteine-mutated sites within

Membrane-associated c-type cytochromes from the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum: purification and characterization of cytochrome c553.

Tetraheme cytochromes involved in photosynthetic electron transport have previously been described associated with the reaction centers of purple photosynthetic bacteria; however, similar heme proteins have not until now been characterized in the phylogenetically distinct green sulfur bacteria. In this paper we describe the first isolation and characterization of a multitheme, membrane-associated cytochrome from a green sulfur bacterium, Chlorobium limicola forma thiosulfatophilum. We show that this cytochrome contains a single polypeptide of 32 kDa apparent molecular mass on SDS-PAGE and has a characteristic broad alpha-band absorption at 553 nm. By both low-temperature absorption and electron paramagnetic resonance spectroscopy, we demonstrate that there are at least four distinct heme groups.

Targeted mutations in the psaC gene of Chlamydomonas reinhardtii: preferential reduction of FB at low temperature is not accompanied by altered electron flow from photosystem I to ferredoxin.

The terminal part of the electron pathway within the photosystem I (PSI) complex includes two [4Fe-4S] centers, FA and FB, which are coordinated by the PsaC subunit. To gain new insights into the electron transfer mechanisms through PsaC, we have generated three mutant strains of the alga Chlamydomonas reinhardtii in which two positively charged residues, K52 and R53, near the FA center have been altered in different ways. The mutations K52S/R53D and K52P/R53D lead to a strong destabilization of PSI. The third mutant K52S/R53A accumulates PSI to 30% of wild-type levels and shares the same residues between two of the cysteine ligands of FA as the PsaC homologue in the green sulfur bacterium Chlorobium limicola, in which FB has a higher redox potential than FA [Nitschke, W., Feiler, U., & Rutherford, A. W. (1990) Biochemistry 29, 3834-3842]. Low-temperature electron paramagnetic resonance (EPR) studies reveal that, in contrast to wild type, FB is preferentially photoreduced in this mutant, as was also observed for C. limicola. The preferential photoreduction of FB could be due to changes in the redox potential of FA and/or to slight structural modifications of the PsaC subunit. However, room temperature optical measurements show that stable charge separation still occurs and, surprisingly, that electron transfer from PSI to ferredoxin proceeds at normal rates in the mutant. As C. limicola, the K52S/R53A and K52S/R53D C. reinhardtii mutants are photosensitive when grown aerobically, but can grow photoautotrophically under anaerobic conditions.

Pl And Epr Spectroscopy Of Point Defects In Detector-Grade Cd1−xZnxTe

AbstractCadmium zinc telluride (CdZnTe) is an emerging material for room-temperature x-ray and gamma ray detectors. The identification and control of point defects and charge compensators are currently important issues. Low-temperature photoluminescence (PL) and electron paramagnetic resonance (EPR) spectroscopies have been used to characterize point defects in CdZnTe crystals grown by the high-pressure Bridgman technique. Luminescence due to shallow donors, shallow acceptors, and deeper acceptors was monitored for a series of samples. An isotropic EPR signal attributed to shallow hydrogenic donors is observed in all samples, and the concentration of shallow donors has been determined. The nature of the defect centers (impurities, vacancies, vacancy-impurity complexes), and the correlation between defect concentration and device performance is discussed.

Neuroprotective effects of hypothermia and U-78517F in cerebral ischemia are due to reducing oxygen-based free radicals: an electron paramagnetic resonance study with gerbils.

Free radicals are implicated as causative agents in various forms of tissue destruction. Considerable circumstantial evidence suggests that oxygen-based free radicals generated as blood flow returns to formerly ischemic brain areas are mainly responsible for the neurodegeneration that follows periods of cerebral ischemia. In general, oxygen-based free radicals are highly reactive and exist for only a brief period of time. This makes the direct measurement of many of these free radicals rather difficult. Much of the current knowledge of free radicals in cerebral ischemia is based on observations of chemical changes brought about by the free radicals rather than on direct observations of the free radicals themselves. Low temperature electron paramagnetic resonance spectroscopy is one method that allows the direct study of free radicals. Compared to samples from sham-operated controls, samples of hippocampus taken from gerbils exposed to 15 min of forebrain ischemia followed by 15 min of reperfusion, frozen in liquid nitrogen less than 20 sec after sacrifice, and scanned by low temperature (100 K) electron paramagnetic resonance, show a significant increase in oxygen-based free radicals and a decrease in carbon-based ubiquinone-like free radicals. The ischemia-induced increase in oxygen-based free radicals is prevented by the intraperitoneal injection of the antioxidant drug U-78517F at the start of reperfusion and by hypothermia. However, neither intervention alters the ischemia-induced reduction in the ubiquinone-like free radicals. This suggests that the neuroprotective actions of hypothermia and U-78517F include a direct reduction in the oxygen-based free radical burden of the post-ischemic tissue.

Corundum-filled resonator systems for high-pressure and low-temperature electron paramagnetic resonance studies

A cylindrical corundum-filled resonator of the TE112-type in the microwave X band, coupled to a coaxial line, is used in two systems designed for the high-pressure, low-temperature electron paramagnetic resonance (EPR) studies. The first one is used up to 800 MPa and over a temperature range of 80–400 K. The second permits EPR measurements down to 4 K in the pressure range below 400 MPa. Pressure and temperature can be simultaneously varied and controlled with the high accuracy needed to study spectral anomalies in the vicinity of the phase transitions in solids.

Active photosynthesis in cyanobacterial mutants with directed modifications in the ligands for two iron-sulfur clusters on the PsaC protein of photosystem I.

The PsaC protein of the Photosystem I (PSI) complex in thylakoid membranes coordinates two [4Fe-4S] clusters, FA and FB. Although it is known that PsaC participates in electron transfer to ferredoxin, the pathway of electrons through this protein is unknown. To elucidate the roles of FA and FB, we created two site-directed mutant strains of the cyanobacterium Anabaena variabilis ATCC 29413. In one mutant, cysteine 13, a ligand for FB was replaced by an aspartic acid (C13D); in the other mutant, cysteine 50, a ligand for FA was modified similarly (C50D). Low-temperature electron paramagnetic resonance studies demonstrated that the C50D mutant has a normal FB center and a modified FA center. In contrast, the C13D strain has normal FA, but failed to reveal any signal from FB. Room-temperature optical studies showed that C13D has only one functional electron acceptor in PsaC, whereas two such acceptors are functional in the C50D and wild-type strains. Although both mutants grow under photoautotrophic conditions, the rate of PSI-mediated electron transfer in C13D under low light levels is about half that of C50D or wild type. These data show that (i) FB is not essential for the assembly of the PsaC protein in PSI and (ii) FB is not absolutely required for electron transfer from the PSI reaction center to ferredoxin.

Production of superoxide from hemoglobin-bound oxygen under hypoxic conditions.

By low temperature electron paramagnetic resonance we have detected the formation of a free radical signal during incubation of partially oxygenated hemoglobin at 235 K. The observed signal has g parallel = 2.0565 and g perpendicular = 2.0043, consistent with the previously reported values for superoxide. The presence of additional EPR signals for oxygen-17 bound hemoglobin, with (017-017)A perpendicular = 63 G and (017-016)A perpendicular = 94 G under identical conditions, confirms the presence of a radical containing two nonequivalent oxygens as required for a superoxide in magnetically inequivalent environments. The superoxide radical has not previously been directly detected during hemoglobin autoxidation because of its rapid dismutation. Our ability to follow the formation of superoxide for more than 15 min is attributed to its production in the hydrophobic heme pocket where dismutation is slow. The enhanced production of this free radical at intermediate oxygen pressures is shown to coincide with enhanced rates of hemoglobin autoxidation for partially oxygenated intermediates. The formation of superoxide in the heme pocket under these conditions is attributed to enhanced heme pocket flexibility. Greater flexibility facilitates distal histidine interactions which destabilize the iron-oxygen bond resulting in the release of superoxide radical into the heme pocket.

An EPR investigation of human methaemoglobin oxidation by hydrogen peroxide: methods to quantify all paramagnetic species observed in the reaction.

The method of Electron Paramagnetic Resonance (EPR) spectroscopy was used to study the reaction of human methaemoglobin (metHb) with hydrogen peroxide. The samples for EPR measurements were rapidly frozen in liquid nitrogen at different times after H2O2 was added at 3- and 10-fold molar excess to 100 microM metHb in 50 mM phosphate buffer, pH 7.4, 37 degrees C. Precautions were taken to remove all catalase from the haemoglobin preparation and no molecular oxygen evolution was detected during the reaction. On addition of H2O2 the EPR signals (-196 degrees C) of both high spin and low spin metHb rapidly decreased and free radicals were formed. The low temperature (-196 degrees C) EPR spectrum of the free radicals formed in the reaction has been deconvoluted into two individual EPR signals, one being an anisotropic signal (g parallel = 2.035 and g perpendicular = 2.0053), and the other an isotropic singlet (g = 2.0042, delta H = 20 G). The former signal was assigned to peroxyl radicals. As the kinetic behaviour of both peroxyl (ROO.) and non-peroxyl (P.) free radicals were similar, we concluded that ROO. radicals are not formed from P. radicals by addition of O2. The time courses for both radicals showed a steady state during the time required for H2O2 to decompose. Once all peroxide was consumed, the radical decayed with a first order rate constant of 1.42 x 10(-3) s-1 (1:3 molar ratio). The level of the steady state was higher and its duration shorter at lower initial concentration of H2O2. The formation of the rhombic Fe(III) non-haem centres with g = 4.35 was found. Their yield was proportional to the H2O2 concentration used and the centres were ascribed to haem degradation products. The reaction was also monitored by EPR spectroscopy at room temperature. The kinetics of the free radicals measured in the reaction mixture at room temperature was similar to that observed when the fast freezing method and EPR measurement at -196 degrees C were used.

An electron paramagnetic resonance study of arthritic joints

Much interest has recently been shown in the detection of reactive oxygen species (ROS) in view of the evidence implicating a primary or secondary role for them in the initiation or progression of many human diseases, the majority of which are characterized by an inflammatory reaction [1-3]. Hydrogen peroxide (H202) and superoxide anion ( 0 2 ) , are produced by diverse cell types and induce free radical-mediated cellular and tissue damage. Redox active transition metal complexes can readily promote the generation of the extremely reactive hydroxyl radical (-OH) from hydrogen peroxide and superoxide [4, 5]. Within the inflamed rheumatoid joint, reactive oxygen species oxidatively modify IgG [6], inactivate alpha-l-proteinase inhibitor [7], peroxidize polyunsaturated lipids [6] and depolymerize hyaluronate [8]. Electron paramagnetic resonance (EPR) spectroscopy has been applied in attempts to assess free radical production in many disease states. For example, in a recent EPR study we have employed the spin trap 3,5,dibromo-4-nitrobenzenesulphonate (DBNBS) to demonstrate reperfusion-induced free radical generation in isolated rheumatoid synovium [9]. Also EPR spectroscopy, usually at low temperatures, has been used to study paramagnetic transition metal complexes in a broad range of biological systems, including enzymes, cells and tissues [1016]. In the present study we have employed lowtemperature EPR spectroscopy to investigate the presence of paramagnetic species in isolated synovium and bone from patients suffering from rheumatoid (RA) and osteo-arthritis (OA). The EPR profiles of samples of normal bone and synovium obtained during ~raumatic injury (TI) surgery were employed as controls. Samples of RA synovium (n = 8), OA synovium ( n = 8 ) , RA cancellous bone ( n = 5 ) and OA cancellous bone (n = 10), from arthritic patients were obtained during total kneeand hip-joint replacement surgery at the Royal London Hospital. Synovium samples ( n = 6 ) were obtained from traumatic injury patients undergoing orthopaedic surgery. All samples were frozen in liquid nitrogen ( -196°C) and stored at 7 0 ° C prior to EPR

Electron paramagnetic resonance spectroscopy as a probe of coordination structure in green heme systems: iron chlorins and iron formylporphyrins reconstituted into myoglobin

A series of ferric low-spin derivatives of myoglobin containing its natural prosthetic group, iron protoporphyrin IX, and reconstituted with iron heme s (a formyl-substituted porphyrin) and iron methylchlorin have been examined using low-temperature electron paramagnetic resonance (EPR) spectroscopy. Good agreement is observed between the EPR properties of parallel derivatives of natural myoglobin and heme s-myoglobin. Likewise, the EPR properties of parallel adducts of three types of iron chlorins, methylchlorin-myoglobin, sulfyomyoglobin (a myoglobin derivative known to contain a chlorin macrocycle) and synthetic chlorin models are similar to each other. The ferric chlorin systems are shown to exhibit increased tetragonality and decreased rhombicity values relative to protoporphyrin/formylporphyrin systems. Thus, EPR spectroscopy is a very useful technique with which to probe the coordination structure of naturally occurring iron chlorin proteins and the method can be used to distinguish between proteins containing iron formylporphyrins and iron chlorin prosthetic groups.

Electron spin resonance of Er3+ in YBiPt.

We report low-temperature electron spin resonance (ESR) measurements in YBiPt, doped with 0.1% Er substituting for Y. The results show that ${\mathrm{Er}}^{3+}$ is located at a cubic lattice site and has a ${\mathrm{\ensuremath{\Gamma}}}_{8}^{(3)}$ as ground state and an overall crystal-field splitting of \ensuremath{\sim}85(20) K. From the ESR spectra we inferred the existence of lattice distortions at the rare-earth site. These results may help in understanding the heavy-fermion system YbBiPt, which has the same structure as YBiPt.

Expression of Desulfovibrio gigas Desulforedoxin in Escherichia coli: PURIFICATION AND CHARACTERIZATION OF MIXED METAL ISOFORMS

The dsr gene from Desulfovibrio gigas encoding the nonheme iron protein desulforedoxin was cloned using the polymerase chain reaction, expressed in Escherichia coli, and purified to homogeneity. The physical and spectroscopic properties of the recombinant protein resemble those observed for the native protein isolated from D. gigas. These include an alpha 2 tertiary structure, the presence of bound iron, and absorbance maxima at 370 and 506 nm in the UV/visible spectrum due to ligand-to-iron charge transfer bands. Low temperature electron paramagnetic resonance studies confirm the presence of a high-spin ferric ion with g values of 7.7, 5.7, 4.1, and 1.8. Interestingly, E. coli produced two forms of desulforedoxin containing iron. One form was identified as a dimer with the metal-binding sites of both subunits occupied by iron while the second form contained equivalent amounts of iron and zinc and represents a dimer with one subunit occupied by iron and the second with zinc.

Purification and Characterization of the Mycobacterium smegmatis Catalase-Peroxidase Involved in Isoniazid Activation

The unique antitubercular activity of isoniazid requires that the drug be oxidized by the katG-encoded mycobacterial catalase-peroxidase to an activated drug form. In order to quantitatively assess the catalytic capabilities of the enzyme, the native catalase-peroxidase from Mycobacterium smegmatis was purified over 200-fold to homogeneity. The enzyme was shown to exhibit both catalase and peroxidase activities, and in the presence of either hydrogen peroxide or t-butyl peroxide, was found to catalyze the oxidation of the reduced pyridine nucleotides, NADH and NADPH, as well as artificial peroxidase substrates, at rates between 2.7 and 20 s-1. The homogeneous enzyme exhibited a visible absorbance spectrum typical of ferric heme-containing catalase-peroxidases, with a Soret maximum at 406 nm. Low temperature (10 K) electron paramagnetic resonance spectra in the presence of ethylene glycol revealed a high spin Fe(III) signal with g values of 5.9 and 5.6. The enzyme was very slowly (t1/2 = approximately 20 min) reduced by dithionite, and the reduced form showed typical spectral changes when either KCN or CO were subsequently added. The M. smegmatis catalase-peroxidase was found to contain 2 heme molecules per tetramer, which were identified as iron protoporphyrin IX by the pyridine hemochromogen assay. The peroxidatic activity was inhibited by KCN, NaN3, isoniazid (isonicotinic acid hydrazide), and its isomer, nicotinic acid hydrazide, but not by 3-amino-1,2,4-triazole. The role of mycobacterial catalase-peroxidases in the oxidative activation of the antitubercular prodrug isoniazid is discussed.