Compelling EPR evidence that the alternative oxidase is a diiron carboxylate protein

The alternative oxidase is a respiratory chain protein found in plants, fungi and some parasites that still remains physically uncharacterised. In this report we present EPR evidence from parallel mode experiments which reveal signals at approximately g=16 in both purified alternative oxidase protein (g=16.9), isolated mitochondrial membranes (g=16.1), and in trypanosomal AOX expressed in Escherichia coli membranes (g=16.4). Such signals are indicative of a dicarboxylate diiron centre at the active site of the enzyme. To our knowledge these data represent the first EPR signals from AOX present in its native environment.     

Evidence for H2O2 as the product of reduction of oxygen by alternative oxidase in mitochondria from potato tubers

Oxygen consumption by alternative oxidase (AOX), present in mitochondria of many angiosperms, is known to be cyanide-resistant in contrast to cytochrome oxidase. Its activity in potato tuber (Solanum tuberosum L.) was induced following chilling treatment at 4 °C. About half of the total O₂ consumption of succinate oxidation in such mitochondria was found to be sensitive to SHAM, a known inhibitor of AOX activity. Addition of catalase to the reaction mixture of AOX during the reaction decreased the rate of SHAM-sensitive oxygen consumption by nearly half, and addition at the end of the reaction released nearly half of the consumed oxygen by AOX, both typical of catalase action on H₂O₂. These findings with catalase suggest that the product of reduction of AOX is H₂O₂ and not H₂O, as previously surmised. In potatoes subjected to chill stress (4 °C) for periods of 3, 5 and ?8 days the activity of AOX in mitochondria increased progressively with a corresponding increase in the AOX protein detected by immunoblot of the protein.     

Low-temperature electron transfer suggests two types of QA in intact photosystem II

The correlation between the reduction of Q_A and the oxidation of Tyr_Z or Car/Chl_Z/Cyt_b559 in spinach PSII enriched membranes induced by visible light at 10 K is studied by using electron paramagnetic resonance spectroscopy. Similar g = 1.95-1.86 Q_A^-•EPR signals are observed in both Mn-depleted and intact samples, and both signals are long lived at low temperatures. The presence of PPBQ significantly diminished the light induced EPR signals from Q_A^-•, Car^+•/Chl^+• and oxidized Cyt_b559, while enhancing the amplitude of the S₁Tyr_Z• EPR signal in the intact PSII sample. The quantification and stability of the g = 1.95-1.86 EPR signal and signals arising from the oxidized Tyr_Z and the side-path electron donors, respectively, indicate that the EPR-detectable g = 1.95-1.86 Q_A^-• signal is only correlated to reaction centers undergoing oxidation of the side-path electron donors (Car/Chl_Z/Cyt_b559), but not of Tyr_Z. These results imply that two types of Q_A^-• probably exist in the intact PSII sample. The structural difference and possible function of the two types of Q_A are discussed.     

pH dependent copper binding properties of a CuA azurin variant with both bridging cysteines replaced with serines

A double mutant of Cu_A azurin was prepared in which both bridging cysteine thiolate ligands of the binuclear Cu_A center were replaced by serine. The copper binding properties of this protein were investigated, and shown to be pH dependent. At lower pH (5.2 ± 0.1), the protein binds one copper per protein molecule as demonstrated by electrospray ionization mass spectrometry. Copper titrations resulted in electronic absorptions at 730 nm (peak) and ca. 330 nm (shoulder) in the UV-Vis spectrum. EPR data show a four line pattern with hyperfine A_∥ = 150 G and g_∥ and g_⊥ values 2.32 and 2.03, characteristic of a type II (T2) copper. Superhyperfines to two nitrogen atoms were also observed. At higher pH (8.5 ± 0.1), the protein binds upto two copper atoms per protein molecule, and copper titrations exhibit a blue transition at 595 nm in the UV-Vis spectrum. The EPR data are consistent with two monomeric sites very similar to one another having hyperfines A_∥ = 182 and 150 G, g_∥ = 2.24 and 2.22 and a similar g_⊥ value of 2.01. These results indicate that both bridging cysteines play a critical role in the Cu_A center, and replacing them with serines is not enough to maintain the symmetrical diamond core structure or the characteristic electronic and functional properties of the Cu_A center.     

Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei

The trypanosome alternative oxidase (TAO) functions in the African trypanosomes as a cytochrome-independent terminal oxidase, which is essential for their survival in the mammalian host and as it does not exist in the mammalian host is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in a haem-deficient Escherichia coli strain has been solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. Analysis of bound iron detected by inductively coupled plasma-mass spectrometer (ICP-MS) reveals a stoichiometry of two bound iron atoms per monomer of rTAO. Confirmation that the rTAO was indeed a diiron protein was obtained by EPR analysis which revealed a signal, in the reduced forms of rTAO, with a g-value of 15. The kinetics of ubiquiol-1 oxidation by purified rTAO showed typical Michaelis-Menten kinetics (K_m of 338 μM and V_max of 601 μmol/min/mg), whereas ubiquinol-2 oxidation showed unusual substrate inhibition. The specific inhibitor, ascofuranone, inhibited the enzyme in a mixed-type inhibition manner with respect to ubiquinol-1.     

Copper complexes with new oxaaza-pendant-armed macrocyclic ligands: X-ray crystal structure of a macrocyclic copper(II) complex

The synthesis of new oxaaza macrocyclic ligands (2-4) derived from O¹,O⁷-bis(2-formylphenyl)-1,4,7-trioxaheptane and functionalized tris(2-aminoethyl)amine are described. Mononuclear copper(II) complexes were isolated in the reaction of the corresponding macrocyclic ligand and copper(II) perchlorate. The structure of the [Cu(2)](ClO₄)₂ complex was determined by X-ray diffraction analysis. The copper(II) ion is five-coordinated by all N₅ donor atoms, efficiently encapsulated by the amine terminal pendant-arm, with a trigonal-bipyramidal geometry. The complexes are further characterized by UV-Vis, IR and EPR studies. The electronic reflectance spectra evidence that the coordination geometry for the Cu(II) complexes is trigonal-bipyramidal with the ligands 1 and 2 or distorted square-pyramidal with the ligands 3 and 4. The electronic spectra in MeCN solutions are different from those in the solid state, which suggest that some structural modification may occur in solution. The EPR spectrum of powder samples of the copper complex with 2 presents axial symmetry with hyperfine split at g_// with the copper nuclei (I = 3/2), which is characteristic of weakly exchange coupled extended systems. The EPR parameters (g_// = 2.230, A_// = 156 × 10⁻⁴ cm⁻¹ and g_⊥ = 2.085) indicate a d_x²-y² ground state. The EPR spectra of the complexes with ligands 3 and 4 show EPR spectra with a poorly resolved hyperfine structure at g_//. In contrast, the complex with ligand 2 shows no hyperfine split and a line shape which was simulated assuming rhombic g-tensor (g₁ = 2.030, g₂ = 2.115 and g₃ = 2.190).     

Synthesis and characterization of a stable paramagnetic hexacoordinated oxochromium(IV) complex with dianionic tetradentate Schiff base ligand salen

A paramagnetic octahedral oxochromium(IV) complex with dianionic tetradentate ligand salen (where H₂salen is N,N′-bis(salicylidene)-1,2-ethylenediamine) has been synthesized. This compound [CrO(OH₂)(salen)] (1) is characterized by elemental analysis, magnetic moment measurement, IR, UV-Vis and EPR spectroscopic studies. Measured room temperature (RT) magnetic moment value is 2.96 BM for 1 indicates a d² system with a triplet ground state. The magnetic moment value rules out a large spin-orbit coupling. The RT and LNT powder EPR spectra of 1 in X-band clearly shows two lines, one around g = 1.965 and the other with larger intensity at g = 4.26 ± 0.10. The first line at g = 1.965 corresponds to the |0> ↔ |±1> transition from the Kramers doublet |±1>, while the broad and intense line at low field with the g-value of 4.26 ± 0.10 is due to the forbidden transition |−1> ↔ |+1>. Compound 1 displays two successive reductions at −0.76 and −1.63 V (versus Ag/AgCl), respectively, while it undergoes only one irreversible oxidation as evident from the well-defined anodic wave at +1.48 V in its cyclic voltammogram.     

Conversion of the g = 4.1 EPR signal to the multiline conformation during the S2 to S3 transition of the oxygen evolving complex of Photosystem II

The oxygen evolving complex of Photosystem II undergoes four light-induced oxidation transitions, S₀-S₁,…,S₃-(S₄)S₀ during its catalytic cycle. The oxidizing equivalents are stored at a (Mn)₄Ca cluster, the site of water oxidation. EPR spectroscopy has yielded valuable information on the S states. S₂ shows a notable heterogeneity with two spectral forms; a g = 2 (S = 1/2) multiline, and a g = 4.1 (S = 5/2) signal. These oscillate in parallel during the period-four cycle. Cyanobacteria show only the multiline signal, but upon advancement to S₃ they exhibit the same characteristic g = 10 (S = 3) absorption with plant preparations, implying that this latter signal results from the multiline configuration. The fate of the g = 4.1 conformation during advancement to S₃ is accordingly unknown. We searched for light-induced transient changes in the EPR spectra at temperatures below and above the half-inhibition temperature for the S₂ to S₃ transition (ca 230 K). We observed that, above about 220 K the g = 4.1 signal converts to a multiline form prior to advancement to S₃. We cannot exclude that the conversion results from visible-light excitation of the Mn cluster itself. The fact however, that the conversion coincides with the onset of the S₂ to S₃ transition, suggests that it is triggered by the charge-separation process, possibly the oxidation of tyr Z and the accompanying proton relocations. It therefore appears that a configuration of (Mn)₄Ca with a low-spin ground state advances to S₃.     

Design of novel iron compounds as potential therapeutic agents against tuberculosis

In the search for new therapeutic tools against tuberculosis two novel iron complexes, [Fe(L-H)₃], with 3-aminoquinoxaline-2-carbonitrile N¹,N⁴-dioxide derivatives (L) as ligands, were synthesized, characterized by a combination of techniques, and in vitro evaluated. Results were compared with those previously reported for two analogous iron complexes of other ligands of the same family of quinoxaline derivatives. In addition, the complexes were studied by cyclic voltammetry and EPR spectroscopy. Cyclic voltammograms of the iron compounds showed several cathodic processes which were attributed to the reduction of the metal center (Fe(III)/Fe(II)) and the coordinated ligand. EPR signals were characteristic of magnetically isolated high-spin Fe(III) in a rhombic environment and arise from transitions between m_S = ± 1/2 (g_eff ~ 9) or m_S = ± 3/2 (g_eff ~ 4.3) states. Mössbauer experiments showed hyperfine parameters that are typical of high-spin Fe(III) ions in a not too distorted environment. The novel complexes showed in vitro growth inhibitory activity on Mycobacterium tuberculosis H₃₇Rv (ATCC 27294), together with very low unspecific cytotoxicity on eukaryotic cells (cultured murine cell line J774). Both complexes showed higher inhibitory effects on M. tuberculosis than the “second-line” therapeutic drugs.     

Studies of the orientation of the mitochondrial redox carriers III. Orientation of the gx and gy axes of the hemes of cytochrome oxidase with respect to the plane of the membrane in oriented membrane multilayers

The EPR absorption properties of the hemes of cytochrome oxidase and their liganded derivatives were examined in oriented multilayers from isolated oxidase, mitochondrial membranes and membrane fragments of a bacterium, Paracoccus denitrificans. The hemes of the oxidase in all the systems investigated were oriented normal to the plane of the multilayers. The directions of the g signals corresponding to the g_x and g_y axes of the g tensor were found to be different in low-spin ferric heme in fully oxidized oxidase and in half-reduced liganded oxidase. It is suggested that this different orientation of g_x and g_y in fully oxidized oxidase and half-reduced liganded oxidase arises because the respective EPR signals belong to two different hemes, those of cytochrome a and a₃.     

Functional characterization of human duodenal cytochrome b (Cybrd1): Redox properties in relation to iron and ascorbate metabolism

Duodenal cytochrome b (Dcytb or Cybrd1) is an iron-regulated protein, highly expressed in the duodenal brush border membrane. It has ferric reductase activity and is believed to play a physiological role in dietary iron absorption. Its sequence identifies it as a member of the cytochrome b₅₆₁ family. A His-tagged construct of human Dcytb was expressed in insect Sf9 cells and purified. Yields of protein were increased by supplementation of the cells with 5-aminolevulinic acid to stimulate heme biosynthesis. Quantitative analysis of the recombinant Dcytb indicated two heme groups per monomer. Site-directed mutagenesis of any of the four conserved histidine residues (His 50, 86, 120 and 159) to alanine resulted in much diminished levels of heme in the purified Dcytb, while mutation of the non-conserved histidine 33 had no effect on the heme content. This indicates that those conserved histidines are heme ligands, and that the protein cannot stably bind heme if any of them is absent. Recombinant Dcytb was reduced by ascorbate under anaerobic conditions, the extent of reduction being 67% of that produced by dithionite. It was readily reoxidized by ferricyanide. EPR spectroscopy showed signals from low-spin ferriheme, consistent with bis-histidine coordination. These comprised a signal at g_max = 3.7 corresponding to a highly anisotropic species, and another at g_max = 3.18; these species are similar to those observed in other cytochromes of the b₅₆₁ family, and were reducible by ascorbate. In addition another signal was observed in some preparations at g_max = 2.95, but this was unreactive with ascorbate. Redox titrations indicated an average midpoint potential for the hemes in Dcytb of + 80 mV ± 30 mV; the data are consistent with either two hemes at the same potential, or differing in potential by up to 60 mV. These results indicate that Dcytb is similar to the ascorbate-reducible cytochrome b₅₆₁ of the adrenal chromaffin granule, though with some differences in midpoint potentials of the hemes.     

Synthesis and characterization of two stable paramagnetic octahedral chromium(IV) complexes with dianionic tridentate SNO donor ligands and of a chromium(III) complex with a ONO donor ligand

Two novel paramagnetic octahedral chromium(IV) complexes with dianionic tridentate SNO donor ligands containing extended π-system have been synthesized while only a paramagnetic octahedral chromium(III) complex is obtained when a related dianionic tridentate ONO donor ligand is used under similar conditions. These bischelate complexes [Cr(abtsal)₂] (1) (abtsalH₂ is the Schiff base of o-aminobenzenethiol and salicylaldehyde), [Cr(4-PhTSCsal)₂] · H₂O (2) (4-PhTSCsalH₂ is the Schiff base of 4-phenylthiosemicarbazide and salicylaldehyde) and K[Cr(sap)₂] · H₂O (3) (sapH₂ is the tridentate Schiff base of salicylaldehyde and o-aminophenol) are characterized by elemental analyses, magnetic moment measurements, IR, UV-Vis and EPR spectroscopic studies. Compound 3 has been structurally characterized by X-ray crystallography. Measured room temperature (RT) magnetic moment values are 2.98 BM for 1 and 2.83 BM for 2 indicating a d² system with a triplet ground state in both the cases. On the other hand, the magnetic moment value for 3 is found to be 3.74 BM at RT and is consistent with the presence of three unpaired electrons for a d³ Cr(III) ion. The magnetic moment values rule out the large spin-orbit coupling which is substantiated by the presence of RT EPR signals. Compounds 1 and 2 exhibit very similar powder EPR spectra at RT and LNT, which show the allowed transition ΔM_s = ±1 (g = 2.004 for both 1 and 2) as well as the “forbidden” half-field transition (ΔM_s = ±2) at g = 4.105 for 1 and g = 4.318 for 2, respectively. The X-band LNT frozen glass EPR spectrum of 1 in DMF shows the presence of zero-field split rhombic symmetry character, and results in the parameters g ≅ 2.0, D = 740 G, and E = 260 G. It suggests that the intensity of ΔM_s = ±2 forbidden transition is large due to the large D value. The X-band frozen glass EPR spectrum of compound 3 in DMF is found to be very similar to that reported for trans-[Cr(py)₄F₂]⁺ in DMF-H₂O-MeOH glass. The large difference (∼700 mV) in the reduction potential for the two octahedral complexes 1 (−1.40 V) and 3 (−0.70 V) is attributed to the difference in their metal ion oxidation states.     

Mixed-valent and radical states of complexes [(bpy)2M(μ-abpy)M′(bpy)2], M,M′ = Ru or Os, abpy = 2,2′-azobispyridine: Electron transfer vs. hole transfer mechanism in azo ligand-bridged complexes

The title complexes were obtained as M^IIM′^II species [(bpy)₂M(μ-abpy)M′(bpy)₂](PF₆)₄, M,M′ = Ru or Os, using the new mononuclear precursor [(bpy)₂Os(abpy)](PF₆)₂ for the osmium-containing dinuclear complexes. One-electron reduction produces radical complexes [(bpy)₂M(μ-abpy)M′(bpy)₂]³⁺ and [(bpy)₂M(abpy)]⁺ with significant contributions from the metals, as evident from the EPR effects on successive replacement of ruthenium by osmium with its much higher spin-orbit coupling constant. The diruthenium and diosmium radical complexes were also studied by EPR at high-frequency (285 GHz), the latter shows an unusually large g anisotropy g₁ − g₃ = 0.25 in frozen solution. Further reduction was monitored by UV/Vis spectroelectrochemistry. Oxidation produced Os^III EPR signals for [(bpy)₂Os(abpy)]³⁺ and [(bpy)₂Os(μ-abpy)Ru(bpy)₂]⁵⁺, indicating a Ru^IIOs^III species for the latter. The diosmium(III,II) and diruthenium(III,II) mixed-valent species remained EPR silent at 4 K, however, they exhibit weak inter-valence charge transfer (IVCT) bands at about 1460 nm. Whereas the cyclic voltammetric response towards reduction is only marginally different for the three dinuclear complexes, successive replacement of ruthenium by osmium causes the first oxidation potential to decrease. The much higher comproportionation constant K_c for the mixed valent diosmium(III,II) state (K_c > 10¹⁵) in comparison to the diruthenium(III,II) analogue with K_c = 10¹⁰ confirms the electron transfer alternative for the valence exchange mechanism, in contrast to the hole transfer established for analogous dinuclear complexes with the formally related diacylhydrazido(2−) bridging ligands.     

Differential Expression of Alternative Oxidase Genes in Soybean Cotyledons during Postgerminative Development

The expression of the alternative oxidase (AOX) was investigated during cotyledon development in soybean (Glycine max [L.] Merr.) seedlings. The total amount of AOX protein increased throughout development, not just in earlier stages as previously thought, and was correlated with the increase in capacity of the alternative pathway. Each AOX isoform (AOX1, AOX2, and AOX3) showed a different developmental trend in mRNA abundance, such that the increase in AOX protein and capacity appears to involve a shift in gene expression from AOX2 to AOX3. As the cotyledons aged, the size of the mitochondrial ubiquinone pool decreased. We discuss how this and other factors may affect the alternative pathway activity that results from the developmental regulation of AOX expression.     

EPR studies of the mitochondrial alternative oxidase. Evidence for a diiron carboxylate center

The alternative oxidase (AOX) is a ubiquinol oxidase found in the mitochondrial respiratory chain of plants as well as some fungi and protists. It has been predicted to contain a coupled diiron center on the basis of a conserved sequence motif consisting of the proposed iron ligands, four glutamate and two histidine residues. However, this prediction has not been experimentally verified. Here we report the high level expression of the Arabidopsis thaliana alternative oxidase AOX1a as a maltose-binding protein fusion in Escherichia coli. Reduction and reoxidation of a sample of isolated E. coli membranes containing the alternative oxidase generated an EPR signal characteristic of a mixed-valent Fe(II)/Fe(III) binuclear iron center. The high anisotropy of the signal, the low value of the g-average tensor, and a small exchange coupling (-J) suggest that the iron center is hydroxo-bridged. A reduced membrane preparation yielded a parallel mode EPR signal with a g-value of about 15. In AOX containing a mutation of a putative glutamate ligand of the diiron center (E222A or E273A) the EPR signals are absent. These data provide evidence for an antiferromagnetic-coupled binuclear iron center, and together with the conserved sequence motif, identify the alternative oxidase as belonging to the growing family of diiron carboxylate proteins. The alternative oxidase is the first integral membrane protein in this family, and adds a new catalytic activity (ubiquinol oxidation) to this group of enzymatically diverse proteins.     

Transition metal complexes of a tridentate ligand bearing two pendant pyridine bases: The X-ray crystal structure of pentacoordinate copper(II) complex

The synthesis of a tridentate ligand, N,N′-bis(2-pyridinyl)-2,6-pyridinedicarboxamide [H₂L] is described together with its manganese(II), cobalt(II), nickel(II), copper(II), zinc(II) and cadmium(II) complexes which were characterized based on elemental analysis, conductivity measurements, spectral, magnetic and thermal studies. The IR spectral studies of all the complexes exhibit a similar feature about the ligating nature of the ligand to the metal ions and revealed that the ligand has coordinated through the nitrogens of the deprotonated amides and the central pyridine. The two pendant pyridine nitrogens in all the complexes are protonated and involved in hydrogen bonding with the oxygens of amide groups. This observation is confirmed by the single-crystal X-ray crystallographic studies of copper(II) complex. The geometry around the copper atom can be viewed as a distorted trigonal bipyramid with τ = 0.74 [structural parameter, τ = (β − α)/60; where α and β are the two basal angles in a five coordinate complex]. The electrochemical study of the copper(II) complex shows single quasi-reversible redox peak [Cu(II) ↔ Cu(I)]. The EPR spectrum of copper(II) complex exhibits rhombic pattern [g₁ = 2.0276, g₂ = 2.0926 and g₃ = 2.18].     

The critical iron-oxygen intermediate in human aromatase

Aromatase (CYP19) is the target of several therapeutics used for breast cancer treatment and catalyzes the three-step conversion of androgens to estrogens, with an unusual C-C cleavage reaction in the third step. To better understand the CYP19 reaction, the oxy-ferrous complex of CYP19 with androstenedione substrate was cryotrapped, characterized by UV-vis spectroscopy, and cryoreduced to generate the next reaction cycle intermediate. EPR analysis revealed that the initial intermediate observed following cryoreduction is the unprotonated g₁ = 2.254 peroxo-ferric intermediate, which is stable up to 180 K. Upon gradual cryoannealing, the low-spin (g₁ = 2.39) product complex is formed, with no evidence for accumulation of the g₁ = 2.30 hydroperoxo-ferric intermediate. The relative stabilization of the peroxo-ferric heme and the lack of observed hydroperoxo-ferric heme distinguish CYP19 from other P450s, suggesting that the proton delivery pathway is more hindered in CYP19 than in most other P450s.     

Synthesis, spectral and magnetical characterization of monomeric [Cu(2-NO2bz)2(nia)2(H2O)2] and structural analysis of similar [Cu(RCOO)2(L-N)2(H2O)2] complexes

A new complex of composition [Cu(2-NO₂bz)₂(nia)₂(H₂O)₂] (1) (nia = nicotinamide, 2-NO₂bz = 2-nitrobenzoate) has been prepared and its composition and stereochemistry as well as coordination mode have been determined by elemental analysis, electronic, infrared and EPR spectroscopy, magnetization measurements over the temperature range 1.8-300 K, and its structure has been solved, as well. The complex structure consists of the centrosymmetric molecules with Cu(II) atom monodentately coordinated by the pair of 2-nitrobenzoato anions and by the pair of nicotinamide molecules, forming nearly tetragonal basal plane, and by a pair of water molecules that complete tetragonal-bipyramidal coordination polyhedron about the copper atom. The complex 1 exhibits magnetic moment μ_eff = 1.86 B.M. at 300 K which decreases to μ_eff = 1.83 B.M. at 1.8 K. The magnetic susceptibility temperature dependence obeys Curie-Weiss law with Curie constant of 0.442 cm³ K mol⁻¹ and with Weiss constant of −1.0 K. EPR spectra at room temperature as well as at 77 K are of axial type with g_⊥ = 2.065 and g_∥ = 2.280 and exhibit clearly, but partially resolved parallel hyperfine splitting with A_II = 160 G, that is consistent with the determined molecular structure of 1. In order to analyze the factors influencing the degree of tetragonal distortion of coordination polyhedron, the dataset of 72 structures similar to that of 1 was extracted from CCD and analyzed. A significant correlation between the average Cu-O_ax bond length and tetragonality parameter τ which was found as a consequence of the Jahn-Teller effect.     

Reductive activation of hexavalent chromium by human lung epithelial cells: generation of Cr(V) and Cr(V)-thiol species

Chromium(VI) compounds (e.g. chromates) are cytotoxic, mutagenic, and potentially carcinogenic. The reduction of Cr(VI) can yield reactive intermediates such as Cr(V) and reactive oxygen species. Bronchial epithelial cells are the primary site of pulmonary exposure to inhaled Cr(VI) and are the primary cells from which Cr(VI)-associated human cancers arise. BEAS-2B cells were used here as a model of normal human bronchial epithelium for studies on the reductive activation of Cr(VI). Cells incubated with Na₂CrO₄ exhibited two Cr(V) ESR signals, g = 1.979 and 1.985, which persisted for at least 1 h. The g = 1.979 signal is similar to that generated in vitro by human microsomes and by proteoliposomes containing P450 reductase and cytochrome b₅. Unlike many cells in culture, these cells continued to express P450 reductase and cytochrome b₅. Studies with the non-selective thiol oxidant diamide indicated that the g = 1.985 signal was thiol-dependent whereas the g = 1.979 signal was not. Pretreatment with phenazine methosulfate eliminated both Cr(V) signals suggesting that Cr(V) generation is largely NAD(P)H-dependent. ESR spectra indicated that a portion of the Cr(VI) was rapidly reduced to Cr(III). Cells incubated with an insoluble chromate, ZnCrO₄, also generated both Cr(V) signals, whereas Cr(V) was not detected with insoluble PbCrO₄. In clonogenic assays, the cells were very sensitive to Na₂CrO₄ and ZnCrO₄, but considerably less sensitive to PbCrO₄.