Reductive Activation Of Cr(Vi) By Nitric Oxide Synthase - American

Center, San Antonio, Texas 78284, and Department of Pediatrics, Center of Integrative .... Free Radical Research Center, Medical College of Wisconsin...
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Chem. Res. Toxicol. 2005, 18, 834-843

Reductive Activation Of Cr(Vi) By Nitric Oxide Synthase Ryan Porter,† Marie Ja´chymova´,‡ Pavel Marta´sek,‡,¶ B. Kalyanaraman,†,§ and Jeannette Va´squez-Vivar*,†,§ Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284, and Department of Pediatrics, Center of Integrative Genomics, First School of Medicine, Charles University, Prague, Czech Republic Received August 11, 2004

Chromium(VI) is a recognized toxicant whose effects have been linked to its reduction to lower oxidation states. Although Cr(VI) is reduced by several systems, it is anticipated that its reduction by nitric oxide synthase (NOS) could have significant effects in endothelial and brain cells that express high constitutive levels of the enzyme. This possibility was examined by electron paramagnetic resonance that showed the formation of a stable Cr(V) species from NOS/Cr(VI). The formation of Cr(V) was calcium/calmodulin-independent indicating that Cr(VI) to Cr(V) reduction occurs at the flavin-containing domain of NOS. Accordingly, Cr(VI) reduction by the reductase domain of NOS and the chimera protein cytochrome-P450reductase+tail-nNOS also generated Cr(V). Activation of tetrahydrobiopterin (BH4)-free NOS with calcium/calmodulin diminished Cr(V) steady-state levels while increasing superoxide formation. Since SOD restored Cr(V) to control levels, this result was taken as evidence for a reaction between Cr(V) and superoxide. Supplementation of NOS with BH4 cofactor not only failed to increase Cr(V) yields but generated superoxide and hydroxyl radical. Since the holoenzyme does not generate superoxide, this reaction indicated that Cr(V) mediates the oxidation of BH4-bound to the enzyme. In the presence of L-arginine, however, Cr(VI) neither enhances superoxide release nor inhibits NO formation from fully active NOS. This suggests that L-arginine protects BH4 from Cr(V)-mediated oxidation. While Cr(V) was inactive toward NO, spin trapping experiments with 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide and oxygen consumption measurements showed that Cr(V) reacts with superoxide by a one-electrontransfer mechanism to generate oxygen and Cr(IV). Thus, reduction of Cr(VI) to Cr(V) by NOS occurs in resting and fully active states. It is likely that the reaction between Cr(V) and superoxide influences the cytotoxic mechanisms of Cr(VI) in cells.

Introduction The noxious effects of air pollution on health have been recognized for several decades. While pulmonary complications such as asthma and bronchitis are documented syndromes associated with air pollution (1-3), stroke and other vascular disorders are also emerging as important outcomes of airborne contaminants (4-8). Current data from the Environmental Protection Agency indicates that more cases of hospitalization and deaths are related to particulate air pollution, with many more events attributable to complications of atherosclerosis and congestive heart failure. Chromium exists in a series of oxidation forms ranging from -2 to +6 valency. From the perspective of human health, the most important species are trivalent -Cr(III)- and hexavalent -Cr(VI)- forms of the metal. Chromium(III) is poorly absorbed and trace amounts are required for normal glucose metabolism. Deficiency in Cr(III) is associated with impaired glucose tolerance, fastening hyperglycemia, glucosuria, elevated body fat, and * Corresponding author. Phone: 414-456-8095; fax: 414-456-6512. † Department of Biophysics, Medical College of Wisconsin. ‡ University of Texas Health Science Center. § Free Radical Research Center, Medical College of Wisconsin. ¶ Charles University.

cardiovascular diseases (9). Conversely, Cr(VI) is a highly hazardous form that causes cancer and other health problems. Cr(VI) is absorbed through the lungs, gut, and skin and is accumulated in tissues more than Cr(III) (10). After absorption, Cr(VI) is reduced to generate Cr(V)/Cr(IV) and Cr(III). In humans, the kidney excretes approximately 60% of an absorbed Cr(VI) dose in the form of Cr(III) within 8 h of ingestion. A small percentage is eliminated in the bile, sweat, and hair. Thus, it has been proposed that differences in bioavailability might in part account for the differences in toxicity of Cr(III) and Cr(VI). Most of the studies on Cr(VI) cytotoxicity thus far have examined the carcinogenic mechanisms of the metal. Emerging studies, however, suggest that air pollution and perhaps Cr(VI) may also have important effects on health. Exposure to particulate pollutant in animal models influence the progression of atherosclerotic lesions, plaque volume, cell turnover, and extracellular lipid pools in coronary and aortic lesions (11). There is evidence that endothelial cell reactivity is influenced by metal ions including Cr(VI) (12-14). Other studies in humans have linked inflammatory activation to pollution-mediated predisposition to atherosclerotic events and other health problems (15-17). This study examines the mechanism of Cr(VI) activation by nitric oxide synthase

10.1021/tx049778e CCC: $30.25 © 2005 American Chemical Society Published on Web 04/08/2005

Chromium(V) Generation by NOS

(NOS)1 and the effects of Cr(VI) exposure on NO production as a potential mechanism by which NOS contributes to Cr(VI) cytotoxicity.

Experimental Procedures Caution: Chromium(VI) is a recognized carcinogen that can be absorbed by the skin and the air. Cautious handling and use of gloves and mask is required all the time to avoid contamination. Reagents. Chromium chloride CrCl3, Cr(III) and potassium dichromate (K2Cr2O7), Cr(VI), desferal, NADPH, L-arginine, calcium chloride, glutathione (GSH), and bovine serum albumin (BSA) were obtained from Sigma Chemical Co. (6R)-5,6,7,8Tetrahydrobiopterin was obtained from Schircks Laboratories (Jona, Switzerland). Bovine brain calmodulin was obtained from Calbiochem. Diethylenetriaminepentaacetic acid (DTPA) was obtained from Fluka Chemika-BioChemika and bovine Cu/ZnSOD (5000 U/mg) and catalase were obtained from Boehringer Mannheim. 5-tert-Butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO) was synthesized and purified as described (18). Ampli Tag Gold polymerase and restriction endonucleases were from Roche, New England Bio Labs and Promega. The pOR 263 vector, containing the rat CYPOR cDNA, was kindly provided by Dr. Charles B. Kasper at McArdle Laboratory for Cancer Research, University of Wisconsin, Madison. The pCWori+ vector was given by Dr. Michael Waterman at Vanderbilt University in Nashville, TN. Expression and Purification of Recombinant Proteins. Neuronal nitric oxide synthase (nNOS) was expressed and purified in the absence of BH4 as previously described (19). The enzyme was purified by HPLC using a Superose G, HR 10/30 column (Pharmacia Biotech AB, Uppsala, Sweden), and the top fractions of the peak corresponding to dimeric enzyme were collected. Enzyme concentration is expressed on the basis of heme content. The reductase domain of rat brain nNOS, including amino acid residues 722-1429, was prepared as described (20). The plasmids for the expression of the chimera protein of NADPH-cytochrome P450 reductase (CPR) containing the 33 amino acids in the C-terminal tails of nNOS (CPR-nNOS)2 in Eschericia coli were constructed from rat CPR wild type in POR263 and the nNOSpCW (21). The 33 amino acids of the C-terminus of nNOS were amplified by PCR from nNOSpCW. The primers for nNOS “tail”: forward with Hinc II site 5′ATATGTVTAACCCTCAGAACGTATGAAGTC 3′ and reverse with Xba I site 5′ AATATVCTAGAGTTAGGAGCTGAAAACCTCATC 3′. Primers were synthesized by the Center for Advanced DNA Technologies at the University of Texas Health Science Center at San Antonio. Protein expression and purification was performed as previously described (21). Electron Paramagnetic Resonance Measurements. Electron paramagnetic resonance (EPR) X-band spectra were acquired at room temperature on a Bruker EMX spectrometer operating at ∼9.3 GHz and 100 kHz field modulation equipped with a standard cavity. Q-band EPR data were acquired on a modified Varian E-110 operating at 34.9 GHz and 100 kHz field modulation. Samples were analyzed in round-fused capillary quartz tubes 0.2 mm (i.d) × 0.33 mm (o.d) (Vitro Dynamics, Inc., NJ). Reactions were initiated by the addition of enzyme to the incubation mixtures with and without 50 mM BMPO in chelextreated Hepes buffer or buffer containing 0.1 mM DTPA. 1 Abbreviations: NOS, nitric oxide synthase; CaM, calmodulin; BH , 4 5,6,7,8-6(R)-tetrahydrobiopterin; FMN, flavin adenine mononucleotide; FAD, flavin adenine dinucleotide; L-Arg, L-arginine; CPR, NADPHcytochrome P450 reductase; SOD, superoxide dismutase; DPI, diphenylene iodonium; BMPO, 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide. 2 Ja ´ chymova´, M. , Marta´sek, P., Panda, S., Roman, L. J., Va´squezVivar, J., Shea, T. M., Ishimura, Y., Kim, J.-J., and Masters, B. S. S. (manuscript in preparation).

Chem. Res. Toxicol., Vol. 18, No. 5, 2005 835 3-Carbamoyl-2,2,5,5-tetramethylpyrrolidine N-oxyl and potassium nitrosodisulfonate (g ) 2.0057) (Aldrich) were used as standards. Biochemical Assays. NOS Activity. Formation of NO from NOS was measured by the hemoglobin assay. Briefly, NOS was added to a reaction mixture containing NADPH (0.1 mM), l-arginine (40 µM), calcium (0.2 mM), calmodulin (20 µg/mL), BSA (0.1 mg/mL), BH4 (10 µM), oxy-hemoglobin (≈8 µM), and chelex-treated Hepes buffer (50 mM, pH 7.4). The rate of oxidation of oxyhemoglobin to methemoglobin was monitored using a double-beam spectrophotometer (Shimadzu UV-2501PC) and quantified by determining the differences in absorbance between 401 and 411 nm per minute at room temperature using an extinction coefficient of 38 mM-1 cm-1. NOS Rd-NOS and CPR-NOS Reductase Activity. The NADPH-driven cytochrome c reductase activity of the enzymes was determined by following changes in absorbance at 550 nm in Hepes buffer pH 50 mM 7.4, containing 200 mM NaCl. NADPH Consumption by nNOS. The initial rates of NADPH oxidation were determined spectrophotometrically at 340 nm. NADPH concentration was calculated using a molar extinction coefficient of 6.22 mM-1cm-1. Reactions were initiated by addition of NADPH (≈0.3 mM) to reaction mixtures (final volume, 0.25 mL) containing NOS in potassium phosphate buffer (0.1 M, pH 7.4), in the presence of Cr(III) or Cr(VI) at the indicated concentrations. Oxygen Uptake. The total oxygen dissolved in Hepes buffer 50 mM, pH 7.4 at 28 °C was measured with an oxygen membrane polarographic detector equipped with a platinum electrode polarized at -600 mV with respect to a silver reference electrode (Rank Brothers Ltd.). The electrode zero readings were adjusted with dithionite.

Results Reductive Activation of Cr(VI) to Cr(V) Species by Nitric Oxide Synthase Reductase Domain. The reduction of Cr(VI) by neuronal NOS, a prototype of constitutive NOS isoforms, was followed by EPR and optical analysis. X-band EPR examination of incubation mixtures containing Cr2O72-, NADPH, and NOS in the resting state, that is, in the absence of Ca2+/calmodulin at room temperature and pH 7.4, led to detection of two stable EPR signals at giso ≈ 1.979 and an anisotropic component at g ) 1.982 with a peak-to-peak line width of approximately 2.2 and 2.7 G, respectively. These signals were attributed to the formation of 52Cr(V) (I ) 0) species (Figure 1, trace A). A quartet of singlets due to 53Cr isotope (I ) 3/2) with Aiso ) 17.30 ( 0.24 was also detected (Figure 1, trace A inset). The Q-band (≈35 GHz) comparison with X-band (≈9.3 GHz) showed a better resolution of the above signals, confirming the formation of at least two different isotropic forms of Cr(V) (Figure 1, trace B): the Cr(V) monomer isotropic component at giso 1.9806 ( 0.0013 and a minor Cr(V) isotropic signal at giso ) 1.9832 ( 0.0002 (Figure 1, trace B inset). Last, a very weak anisotropic component with g// ≈ 1.8 and g⊥ ≈ 1.977 was also evident. No EPR signals were detected in the absence of the enzyme or the metal (not shown). Changes in Cr(VI) absorbance (λmax ) 372 nm, pH 7.4) in incubation mixtures of Cr2O72- with NOS were followed with time (Figure 2, panel A). The decrease in Cr(VI) absorbance over time was evident, but it was not accompanied by the formation of UV-vis active products in the 300-800 nm range (Figure 2, panel A). Previous studies have shown that the reduction of Cr(VI) by ascorbate generates an intermediate with a strong absorption at 582 nm, which has been attributed to Cr(V) (22). Although the EPR data showed that Cr(V) is readily

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Figure 1. X-band and Q-band EPR spectra of the Cr(V) species generated from BH4-free NOS dependent reduction of Cr(VI). The spectra were recorded at room temperature from incubation mixtures containing nNOS (6 µg), NADPH (0.1 mM), and Cr2O72- (0.1 mM) in Hepes buffer 50 mM, pH 7.4. The spectra were recorded after 2 min (X-band) and 5 min (Q-band) of reaction that was initiated by addition of enzyme. (A) X-band EPR shows a strong singlet (4) with giso = 1.979 using the following instrumental conditions: microwave power, 10 mW; modulation amplitude, 1 G; scan rate, 10 G/s; number of scans, 10. The inset shows an expended EPR spectrum showing (b): 53Cr (I ) 3/2) A ) 17.30 ( 0.24 G and (‡): suspected Cr(V) iso anisotropic component g// = 1.982 and g⊥ = 1.977. Instrumental conditions were the same except scan rate was 3.5 G/s. (B) Q-band EPR spectrum showing a better resolved Cr(V) spectra of the isotropic Cr(V) species (4) giso ) 1.9806 ( 0.00013 and a minor isotropic component (‡) giso ) 1.9832 ( 0.0002. The inset shows the spectra in a 10× increase scale, which reveals the presence of an additional Cr(V) anisotropic component (O) g// = 1.80 and g⊥ = 1.977. Instrumental conditions: microwave power, 5 mW; modulation amplitude, 2.5 G; scan rate, 3.3 G/s; number of scans, 5.

generated and stable, under the experimental conditions used in this assay, there was no evidence for the formation of products at ∼580 nm. As shown in Figure 2 (panel C), Cr(VI) consumption was slow with an estimated rate of disappearance of approximately 0.098 nmoles min-1 or 50 nmoles min-1 mg protein-1. Although it is possible that Cr(V) levels are too low to be detected by light absorption, similar results were obtained after increasing Cr(VI) or NOS. The EPR and UV-vis data indicated that Cr(VI) is reduced at the flavo-containing carboxy-reductase domain of NOS, that is, reactions are catalyzed by the enzyme in the resting state. This mechanism was further examined by following the reduction of Cr(VI) by the reductase domain of NOS (Rd-NOS). As shown in Figure 2 (panel B), consumption of both Cr(VI) and NADPH by the RDNOS was evident and was inhibited by the flavoprotein inhibitor, diphenylene iodonium (Figure 2, panel B, inset). This strongly suggested that reduction of Cr(VI)

Figure 2. UV-vis measurement of Cr(VI) consumption by fulllength NOS, reductase domain of NOS, and CPR-nNOStail chimeric protein. (A) Reactions were initiated by addition of 0.13 µM of the NOS incubation mixtures of 100 µM Cr(VI), NADPH in Hepes buffer 50 mM, pH 7.4, at room temperature. Measurements of Cr(VI) (372 nm) (peak 1) were blanked against NADPH (340 nm) (peak 2) and vice versa. Inset: Same as above except reaction was initiated by addition of 0.13 µM CPR-NOS. Scans were performed at 1.5 interval. (B) Same as A except 0.13 µM Rd-NOS was used. Inset: inhibition of NADPH and Cr(VI) consumption by DPI (0.1 mM). (C) Cr(VI) consumption was calculated from 372-nm absorbance (peak 1) over time for ([) full-length NOS (]) reductase domain of NOS and (9) for CPRNOS. Every measurement was repeated at least twice showing an error of less than 0.5%.

Chromium(V) Generation by NOS

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Figure 3. Quantification of Cr(V) formation from the reduction of Cr(VI) by NOS and related proteins. Cr(V) concentrations were calculated by double integration of the EPR spectrum obtained in incubations of Cr(VI) with ([) 0.12 µM NOS, (]) 0.13 µM RDNOS, and (9) CPR-NOS, respectively, in 50 mM Hepes buffer pH 7.4, at room temperature using 3-carboxy-2,2,5,5-tetramethylpyrrolidine-N-oxyl as standard. (Insets) Cr(V) spectrum detected in incubations of 100 µM Cr(VI), 0.12 µM NOS, and 0.13 µM or 0.2 µM CPR-NOS in 50 mM Hepes buffer, pH 7.4, at room temperature. Instrumental conditions: 10 mW, modulation amplitude 1 G, scan rate 10 G/s, number of scans 5.

by Rd-NOS occurs through the formation of an electrontransfer complex between reduced flavins cofactor and Cr(VI). The reduction of Cr(VI) by Rd-NOS, however, was much slower than full-length enzyme at a same enzyme concentration. A possible explanation for the difference is that Rd-NOS presents approximately 50% lower NADPH-cytochrome c reductase activity than fulllength NOS (2485 ( 35 versus 5123 ( 82 min-1, respectively). To further examine the correlation between reductase activity and rates of Cr(VI) reduction, the kinetics of reaction was examined using another flavoprotein that has high reductase activity. This is the chimera protein of NADPH-cytochrome P450 reductase (CPR) and the 33 amino acids of the “tail” C-terminal of nNOS (CPR-NOS)1 (21). The CPR-NOS has a cytochrome c that has a reductase activity >7-fold higher than full-length NOS (unpublished results) (23). In this case, however, the rates of Cr(VI) consumption by CPRNOS, at the same protein concentrations used in NOS assays, were approximately the same seen with fulllength NOS, that is, 0.102 nmoles min-1. Thus, the increased reductase activity of CYPOR did not translate into higher rates of Cr(VI) reduction. The reduction of Cr(VI) by Rd-NOS CPR-NOS produced Cr(V) monomeric species with a giso ) 1.980 (Figure 3). Even though the g-value of the Cr(V) was almost identical to that generated by NOS, the Cr(V) signal generated by CPR-NOS was less steady. This was shown in the quantification of the Cr(V) signal, which demonstrated significantly lower Cr(V) concentrations and CPR-NOS incubations (Figure 3). This result suggests that, despite the fact that CPR-NOS reduces Cr(VI) at rates comparable with NOS, the Cr(V) decays faster in the CPR-NOS system. A possible mechanism that could explain the low Cr(V) yields is that CPR-NOS further reduces Cr(V) to lower oxidation states and hence dimin-

ishes Cr(V) steady-state levels. This mechanism is supported by the higher NADPH consumption observed in CPR-NOS system compared to NOS (see Figure 2, peak 2 in the inset). In the case of Rd-NOS, however, the low Cr(V) yields correlate better with the reduced rate of Cr(VI) reduction compared to full-length NOS. The effect of metal chelators desferal and diethylenetetraminopentaacetic acid (DTPA) on Cr(V) yields in NOS incubations was also examined. As shown in Figure 4 (panel A), the EPR intensity of Cr(V) was significantly reduced by desferal but not by DTPA suggesting that desferal interferes with Cr(VI) reduction. Indeed, UVvis data of Cr(VI) consumption demonstrated that desferal increases the rates of Cr(VI) consumption by NOS (Figure 4, panel B). Parallel experiments showed that the reaction between Cr(VI) and desferal is negligible. Also, there was no differences in the rates of Cr(VI) consumption in the presence of desferal 0.5 or 1 mM (not shown). Thus, it is likely that desferal directly contributes to Cr(VI) reduction to lower oxidation states by reacting with Cr(V). Overall, these results demonstrate that metal chelators, DTPA and desferal, do not inhibit Cr(VI) reduction by NOS and strongly support the mechanism of Cr(VI) reduction by the flavin domain of NOS. As expected for an intermediate species, Cr(V) steadystate levels are dependent not only on the rates of Cr(VI) reduction but also on their stabilization. Although Cr(VI)/dehydroascorbate generated higher Cr(V) levels than Cr(VI)/NOS, this difference does not entirely explain the lack of spectrophotometric evidence for Cr(V) formation. Electronic absorption spectra for stable Cr(V) complexes with 2-hydroxy acids show activity in the 500750 nm range. In the same region, however, a strong absorption due to Cr(III) complex with R-aminoisobutyric acid has been reported (24). These observations indicate that light absorption in the 500-650 nm is not a unique

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Figure 4. Effect of metal chelators on the reduction of Cr(VI) by NOS. (A) EPR measurements of Cr(V) yield in incubations of Cr2O7-2 (100 µM) and nNOS (4 µg), NADPH (150 µM) in phosphate buffer 0.1 mM, pH 7.4, with and without DTPA (0.1 mM) or desferal (0.1 mM). (B) UV-vis measurements of Cr(VI) consumption in incubations of Cr2O7-2 (100 µM) and NOS (12 µg), NADPH (150 µM) in phosphate buffer 0.1 mM, pH 7.4. Readings were blanked against NADPH. (Inset) Same incubation as above plus 0.5 mM desferal.

Porter et al.

Figure 5. NOS-mediated Cr(V) formation. (A) Incubation mixtures of Cr2O72- (100 µM) with nNOS (4 µg), NADPH (150 µM) in phosphate buffer 0.1 mM, pH 7.4, and CaCl2 (0.2 mM); (B) as A plus calmodulin (20 µg/mL); (C) as B plus SOD (10 µg/mL); (D) as B plus catalase (10 µg/mL); (E) as B without Cr2O72-; (F) as B without nNOS. Instrumental conditions: microwave power, 20 mW; modulation amplitude, 2.5 G; frequency, 9.861 Hz; gain, 105; number of scans, 4.

Some of the potential reactions by which Cr(V) turnover could be accelerated are shown in eqs 1-3 (26-29).

Cr(V) + O2•- f Cr(IV) + O2

(1)

Cr(V) + H2O2 f Cr(VI) + •OH + OH-

(2)

NOS

feature for Cr(V) complexes and that the lack of evidence for product formation in Cr(VI)/NOS could be taken as an indication of Cr(III) nonappearance. However, the last possibility needs to be confirmed by low-temperature EPR. The Effect of Ca2+/Calmodulin on NOS-Mediated Cr(VI) Reduction. Activation of the electron flow from the reductase to the oxygenase domain of NOS with Ca2+/ calmodulin (CaM) is accompanied by increased rates of electron transfer between the flavins. As a consequence, a higher rate of reduction of electron acceptors such as ferricyanide and cytochrome c (24, 25) is observed. In the Cr(VI)/NOS system, however, enzyme activation did not increase Cr(V) yields compared to those generated by the resting enzyme (Figure 5 compare trace A with trace B). Actually, the Cr(V) signal was diminished following enzyme activation suggesting that either Cr(VI) is not a good substrate for the active enzyme or that Cr(V) species turnover is accelerated as a consequence of enzyme activation. Addition of SOD, but not catalase, increased Cr(V) yield generated by active NOS (Figure 5, trace C and D).

Cr(V) + 1/2NADPH 98 Cr(IV) + 1/2 NADP+ (3) Equations 1 and 2 implicate superoxide anion radical and hydroxyl radical, whose formation was inferred by the effects of SOD and catalase in the experiments above. To examine the formation of oxygen radical species in Cr(VI)/NOS system, the BMPO spin trap was used. As shown in Figure 6 (trace A), BMPO-superoxide radical adduct (BMPO-OOH) was detected upon activation of NOS with Ca2+/CaM alone. The addition of Cr(VI) resulted in the codetection of a Cr(V) signal and a composite spectrum of BMPO adducts that can be simulated considering 73% contribution of BMPO-OOH (two isomers), 23.5% BMPO-hydroxyl radical (BMPOOH), and 3.5% of BMPO-carbon-centered radical of unknown origin. The yields of BMPO-OOH were lower than that detected in NOS alone (Figure 6, trace B). In the absence of Ca2+/CaM, superoxide was barely detected, while Cr(V) signal intensity reached its highest intensity (Figure 6, trace C). No Cr(V) signal or BMPO-OOH was detected in the absence of NOS (Figure 6, trace D). These data confirmed the results obtained by direct EPR (Figure 1 and Figure 5), showing that NOS is able to

Chromium(V) Generation by NOS

Figure 6. Superoxide and Cr(V) generation from incubations of Cr(VI) with NOS. (A) (solid line) Incubation mixtures containing BH4-free nNOS (4 µg), CaCl (0.2 mM), calmodulin (20 µg/ mL), BMPO (50 mM) in phosphate buffer 0.1 M, pH 7.4; (dotted lines) computer simulation fitted considering two isomers of BMPO-OOH: {58%} aN ) 13.25 G, aH ) 11.89 G; {42%} aN ) 13.22 G, aH ) 9.58. (B) (solid line) As A plus Cr2O72- (100 µM); (dotted lines) computer simulation fitted considering two isomers of BMPO-OOH: {38%} aN ) 13.16 G, aH ) 9.35 G; {35%} aN ) 13.25 G, aH ) 11.55 G; BMPO-OH: {23.5%} aN ) 13.25 G, aH ) 14.01 G; aH ) 0.66 G; BMPO-carbon radical: {3.5%} aN ) 14.65 G, aH ) 20.95 G. (C) As A without calmodulin. (D) As A without NOS. Instrumental conditions: microwave power, 20 mW; modulation amplitude, 1G; gain, 105; number of scans, 4.

reduce Cr(VI) to Cr(V) in a Ca2+/CaM-independent mechanism. They also indicated that the reduction of Cr(VI) does not inhibit superoxide formation from NOS upon activation with Ca2+/CaM. Finally, they show that both superoxide and hydroxyl radicals are generated concomitant to Cr(VI) reduction by NOS. Superoxide release from the oxygenase domain of NOS is Ca2+/CaM-dependent and inhibited by tetrahydrobiopterin (BH4) cofactor (26). Thus, to distinguish between superoxide generated from NOS and from the redox reactions of Cr(VI), the effects of BH4 in NOS/Cr(VI)derived superoxide were examined. As shown in Figure 7 (trace A-D), BH4 diminished superoxide release from NOS in a concentration-dependent fashion. At a concentration of 1 µM BH4, superoxide detection was negligible. Addition of SOD (10 µg/mL) completely abolished BMPOOOH indicating the trapping of free superoxide anion radical (not shown). In incubations of NOS with Cr(VI) (Figure 7, trace E-H), BH4 decreased superoxide although to a lesser extent than in the absence of chromium ion. A composite spectrum of BMPO-OOH (59%), BMPOOH (23%), and BMPO-carbon-centered radical (18%) was detected (Figure 7, trace G). This indicated that Cr(VI) activation by NOS is accompanied by increased superoxide formation as compared to incubation with enzyme alone (trace D vs trace G). In incubations with SOD, the detection of superoxide and hydroxyl radical was marginal whereas Cr(V) steady-state levels were notably increased. (Figure 7, trace H). Similar effects of SOD on Cr(V) yields were shown in the direct EPR experiments (Figure 5). Together these results indicate

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Figure 7. BH4 effects on superoxide release from BH4-free NOS (left) and BH4-free NOS/Cr(VI) (right). Incubation mixtures contained: (A) nNOS (4 µg protein), calcium (0.2 mM), calmodulin (20 µg/mL), and BMPO (50 mM) in phosphate buffer, pH 7.4; (dotted lines) computer simulation fitted considering two isomers of BMPO-OOH: {57%) aN ) 13.25 G, aH ) 11.91 G; {43%} aN ) 13.25 G, aH ) 9.65 G; (B) as A plus 0.01 µM BH4; (C) as A plus 0.1 µM BH4. (dotted line) Computer simulation fitted considering two isomers of BMPO-OOH: {49.5%) aN ) 13.25 G, aH ) 11.1 G; {50.5%} aN ) 13.22 G, aH ) 9.86 G; (D) as A plus 1 µM BH4; (E) as A plus K2Cr2O7 (0.25 mM). (dotted line) Computer simulation fitted considering: BMPO-OOH: {44.5%} aN ) 13.25 G, aH ) 11.95 G; {31%} aN ) 13.2 G, aH ) 9.71 G; BMPO-OH: {20%} aN ) 13.06 G, aH ) 14.5 G, aH ) 0.94 G, BMPO-carbon radical: {4.5%} aN) 14.8 G, aH ) 20.0 G; (F) as E without calmodulin plus BH4 (1 µM); (G) as E plus BH4 (1 µM). (dotted line) Computer simulation fitted considering: BMPO-OH: {23%} aN ) 13.0 G, aH ) 14.3 G; BMPOOOH {59%} aN ) 13.9 G, aH ) 12.65 G; BMPO-carbon radical: {18%} aN ) 14.89 G, aH ) 20.63 G; (H) as E plus SOD 10 µg/ mL. Instrumental conditions were the same as in Figure 5.

that Cr(VI) is reduced by Ca2+/CaM-activated NOS generating Cr(V) and reactive oxygen species and that the reaction between Cr(V) and superoxide decreases Cr(V) steady-state levels. The experiments above, however, did not exclude the possibility that activation of NOS increases the rates of Cr(V) reduction to Cr(IV) (eq 3), which is anticipated to alter NOS activity. Thus, the effects of Cr(VI) redox activation in NOS activity were examined by following both NADPH consumption and NO formation. The NADPH consumption by NOS in the resting state was stimulated by Cr(VI) in a concentration-dependent manner (Figure 8, panel A). Changes in NADPH consumption, however, did not directly correlate with Cr(VI) concentrations. To examine if Cr(III), an anticipated product from Cr(VI)/NOS reactions, interfered with NADPH consumption, CrCl3 was added to NOS incubation mixtures. However, Cr(III) did not alter NADPH consumption (Figure 8, panel A inset) indicating that Cr(III) formation does not contribute to the nonlinear relationship between changes in NADPH consumption and Cr(VI) concentrations. Another influencing factor in the kinetics of NADPH consumption is due to constraints in Cr(VI) binding to NOS. As mentioned above, activation of NOS increases electron-transfer rates between flavins (FMN, FAD) at the reductase domain of NOS, which increases not only NADPH consumption severalfold but also enhances the reduction of electron acceptors such

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Figure 8. Cr(VI)-increases NADPH consumption rates by NOS. Incubations containing (A) nNOS (2 µg) and increasing concentrations of Cr2O72- in phosphate buffer 0.1 M, pH 7.4. (Inset) nNOS (2 µg) and CrCl3. (B) As A plus CaCl2 (0.2 mM) and calmodulin (20 µg/mL) and BH4 (10 µM) and L-arginine (0.1 mM). (C) NO formation was determined by the hemoglobin assay as described in Materials and Methods.

as cytochrome c and ferricyanide. However, enzyme activation did not increase Cr(VI)-stimulated NADPH consumption (Figure 8, panel B). This result suggested that Cr(VI) has a lower affinity for NOS than ferricyanide. This mechanism is also consistent with the lack of Cr(VI) effects on NOS activity as demonstrated by measuring NO formation with the hemoglobin assay. As shown in Figure 8 (panel C), no quantifiable changes in NO formation were seen in incubations of NOS with Cr(VI). Although these results cannot exclude the possibility that NOS reduces Cr(V) to Cr(IV) (eq 3), they certainly indicate that the reaction is slower than L-arginine conversion to NO (Figure 8, panel C). Cr(V) Formation by NO-Generating NOS. Formation of Cr(V) by NOS, under the same conditions used to measure NO production, was followed by EPR to examine the possibility that NO formation inhibits the reduction of Cr(VI). It was shown that fully active NOS, producing NO at a rate of 431.5 ( 8.7 nmoles min-1 mg protein-1, generated Cr(V) concentrations comparable to those detected with NOS in the resting state (Figure 9). Therefore, NO formation from NOS does not preclude Cr(VI) reduction by the enzyme. Under these conditions, superoxide radical generation was marginal (Figure 9, panel B), suggesting that fully active NOS is less sensi-

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tive to Cr(VI)-effects than BH4-saturated NOS as shown above (compare Figure 9, trace G with Figure 9, panel B). Since superoxide formation is inhibited by BH4 and L-arginine, it is possible that L-arginine protects BH4 from Cr(VI)-mediated oxidation but does not prevent the reduction of the metal by NOS. Lower Cr(V) levels were detected in incubations of fully active NOS with LNAME. However, upon addition of the BMPO, the Cr(V) returned to controls levels. This result suggested that L-NAME increases superoxide formation from nNOS with concomitant decrease in Cr(V) levels. This mechanism was confirmed by the protective effects of SOD on Cr(V) levels in L-NAME incubations (Figure 9, panel C and D). Together these results indicated that Cr(V) levels are not affected by NO production; however, they are decreased by reacting with superoxide. The Reaction of Cr(V) with Superoxide. The reaction in eq 1 {Cr(V) + O2•- f Cr(IV) + O2} predicts that oxygen evolves from incubations where Cr(V) is generated concomitantly to superoxide. This mechanism was tested by measuring the oxygen consumption in Cr(VI)/NOS incubation mixtures. Resting NOS consumed oxygen at a rate of 44.5 ( 2.9 nmoles/min/mg protein, which was marginally affected by the addition of Cr(VI) (Figure 10, trace A). Upon activation of NOS with Ca2+/ CaM, the rate of oxygen uptake was 1439 ( 228 nmoles/ min/mg protein (Figure 10, trace B); this is a >30-fold increase. The addition of Cr(VI) after the rapid phase of oxygen consumption did not further stimulate oxygen uptake. Conversely, SOD and catalase stimulated oxygen evolution. SOD restored approximately 27.1% and catalase 23.5% of the total amount of oxygen consumed by NOS (Figure 10, trace B). When Cr(VI) was present from the beginning of the experiments (Figure 10, trace C), there was a 27% decrease in the rates of oxygen consumption and approximately a 46% decrease in the total oxygen uptake. The addition of SOD, but not catalase, restored approximately 22% of the total oxygen consumed (Figure 10, trace C). This evidence was taken as demonstration that hydrogen peroxide from superoxide dismutation is not accumulated in the system. This result is expected because the Cr(V), generated from Cr(VI) reduction at the reductase domain of NOS, reacts with superoxide, generated from the heme-oxygenase domain of NOS, to generate oxygen and Cr(IV). The overall effect of Cr(VI) in NOS incubation mixtures is therefore to decrease the amount of oxygen consumed and the accumulation of reactive oxygen species. This mechanism was further tested by comparing the effects of SOD in the above incubation mixture. As shown in Figure 10 (trace D), the rate and total oxygen consumed were decreased indicating the evolution of oxygen in the system. In this case, however, accumulation of hydrogen peroxide was higher than in the Cr(VI)/NOS system that does not accumulate hydrogen peroxide. It is likely that Cr(V) cannot fully outcompete the reaction between SOD and superoxide, and thus some of the superoxide is converted into hydrogen peroxide.

Discussion The major conclusions of this study are (1) NOS reduces Cr(VI) to Cr(V) in a calcium/calmodulin-independent mechanism at the reductase domain; (2) Cr(VI) stimulates superoxide formation from BH4-saturated NOS; and (3) Cr(V) reacts with superoxide to generate

Chromium(V) Generation by NOS

Chem. Res. Toxicol., Vol. 18, No. 5, 2005 841

Figure 9. Formation of Cr(V) by NOS under resting and NO-generating conditions. Incubation mixtures contained (A) nNOS (4 µg) and the following additions in sequential additive order: NADPH (0.1 mM), BMPO (25 mM), K2Cr2O7 (100 µM). (B) nNOS (4 µg), calcium (0.2 mM), calmodulin (20 µg/mL), BH4 (10 µM), L-arginine (0.1 mM), and BMPO (50 mM), K2Cr2O7 (100 µM) in phosphate buffer pH 7.4. (C) As B in the presence of L-NAME (0.5 mM). (D) As C in the presence of SOD (10 µg/mL).

Figure 10. Cr(VI) decreases oxygen consumption and reactive oxygen formation by NOS. Incubations were performed at 28 °C in 50 mM Hepes buffer, pH 7.4. Reactions were initiated by the addition of nNOS (20 µg) to incubation mixtures containing (A) NADPH (0.1 mM); (B) as A plus calcium (0.2 mM), calmodulin (20 µg/mL); (C) as B plus K2Cr2O7 (100 µM), and (D) as C plus SOD (10 µg/mL). Other additions of Cr(VI) (100 µM), SOD (10 µg/mL), and catalase (10 µg/mL) were at the indicated time points.

oxygen and to diminish reactive oxygen species formation.

Nitric oxide synthase is a prototype of FAD- and FMNrequiring enzymes with an extraordinary ability to reduce electron acceptors and to bind metals including iron, zinc, nickel, and so forth. (27, 28). Here, it is shown that NOS mediates the reduction of Cr(VI) to Cr(V) by a calcium/calmodulin-independent mechanism indicating that the reaction occurs at the reductase domain of the enzyme. The detected Cr(V) species giso ) 1.98 is identical to that generated by other reducing systems including ascorbate (29), oxygen-rich polyfunctional ligands (30), and thiol compounds. This species was attributed to formation of monomeric Cr(V), which was notably EPR persistent. The Cr(V) with a giso ) 1.982 is likely due to Cr(V) five-coordinated species (31) while the minor anisotropic component appears to be a dimeric Cr(V) species presenting angular distortion (32). The reduction of Cr(VI) at the flavin-portion of NOS was also indicated by studies performed with Rd-NOS and CPR-NOS. Both proteins reduced Cr(VI) at rates that appear unrelated to their cytochrome c reductase activity to generate mostly Cr(V) giso ) 1.98. The fact that the flavoproteininhibitor, DPI, blocked the reduction of Cr(VI) strongly indicates that NOS catalyzes the reaction. The involvement of other redox active metals such as iron in our assay conditions appears unlikely since the metal chelators DTPA and desferal do not alter Cr(VI) reduction. Unlike the reaction of Cr(VI) with other flavoproteins (33), the Cr(V) generation by NOS in the resting state was followed neither by increased superoxide nor hydroxyl radical formation. Therefore, superoxide anion radical and hydrogen peroxide, but not Cr(V)/Cr(IV), became limiting reagents for hydroxyl radical production from Cr(VI)/NOS in the resting state. The limited reac-

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Scheme 1. Cr(VI) Reduction to Cr(V) at the Reductase Domain of NOS in the Resting, Ca2+/ CaM-Active and Fully Active NO-Generating States

tive oxygen species formation is further indicated by comparing the rates of NADPH consumption with Cr(V) generation. The NADPH consumption by Cr(VI)/NOS occurred at an approximate rate of 0.44 ( 0.02 µM/min. In the case that all of the NADPH is utilized to reduce Cr(VI), then the theoretical rate of Cr(V) would be close to 0.8 µM/min, which is in close agreement with the estimated rates of Cr(V) generation of 0.5 µM/min. Therefore, the Cr(V) generation at g ) 1.98 accounts for >65% NADPH consumed by NOS/Cr(VI) system. Activation of BH4-free NOS with calcium/calmodulin diminished Cr(V) levels, despite the Cr(V)-stimulated NADPH consumption by NOS. This result suggested that Cr(V) could be lost through the reaction with superoxide anion radical, which is released from the heme-oxygenase domain of NOS (27) (Scheme 1). This was verified by the effects of SOD that restored Cr(V) levels back to control levels. To further verify that Cr(V) decays by reacting with superoxide, Cr(V) yields were determined in NOS supplemented with BH4, which inhibits superoxide release from the enzyme to augment hydrogen peroxide (26). Under these conditions, however, Cr(V) levels did not increase. Instead, hydroxyl and superoxide anion radicals were detected. Since Cr(V) itself does not generate reactive oxygen species, this reaction is likely to involve Cr(V) and hydrogen peroxide, released from the heme group at the oxygenase domain of NOS, thereby causing the product of the reaction, hydroxyl radical, to oxidize BH4 bound to the NOS protein and to facilitate NOS uncoupling and the consequent generation of superoxide. It is also possible that Cr(V) directly oxidizes BH4 stimulating superoxide formation from NOS. The mechanism above anticipated that Cr(VI) inhibits nitric oxide formation from NOS. However, activity assays and spin trapping experiments showed that this is not the case. Therefore, NO production from NOS is critical to prevent BH4 degradation and hence the propagation of oxidative reactions initiated by Cr(V).

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Since Cr(V) levels are not affected by activation of NO formation, the possibility that Cr(V) directly oxidizes the BH4 in NOS can be dismissed. The apparent resistance of NOS-bound BH4 to oxidation is a recurrent observation in the literature, which may indicate a role for L-arginine. Here, we show that L-arginine inhibits Cr(VI) reactions by protecting BH4 from oxidation. Thus, the reduction of Cr(VI) by NOS under turnover conditions, that is, generating NO, is not accompanied by superoxide formation (Scheme 1). As a corollary, this mechanism indicates that Cr(VI)/NOS does not generate peroxynitrite, although it does not exclude the possibility that NO generated from Cr(V)/NOS could react with superoxide from other sources to generate peroxynitrite, which could contribute to the cytotoxic effects of Cr(VI). However, the reaction between Cr(V) and superoxide, to generate Cr(IV) and oxygen, is an important reaction that likely limits the oxidative damage associated to Cr(V) formation. While most of the studies have implicated Cr(V) in increasing oxidative stress, this concept may not apply to systems generating superoxide because Cr(V) by oxidizing superoxide to oxygen reduces the levels of hydrogen peroxide and consequently hydroxyl radical and peroxynitrite formation. This mechanism supports the idea that metal toxicity is directly mediated by the balance between Cr(V) and Cr(IV) redox forms (34, 35). Endothelial cells may be a preferential target for Cr(VI) cytotoxicity. This is indicated by the ability of NOS, an enzyme constitutively expressed in these cells in high levels, to catalyze the reductive activation of Cr(VI). Also, because of their location, endothelial cells serve as an important physical barrier for Cr(VI) contamination through airways. Assuming that the uptake of Cr(VI) by endothelial cells is comparable to other cells, the existence of an efficient system to generate Cr(V) will likely make endothelial cells more prone to Cr(VI) cytotoxic effects. It is anticipated that NOS will enhance the formation of Cr(V)-thiol and Cr(VI)-peptide interaction by increasing Cr(V) availability and indirectly supporting Cr(V)-ligand-exchange reactions (36). For instance, by augmenting Cr(V) thiol complexes, Cr(V) may affect signaling pathways involving thiol groups in these cells. These complexes may play a key role in activating inflammatory-like responses, like the increased cell adhesion receptor expression seen in Cr(VI)-treated endothelial cells. The consequences of such a cascade of events clearly could be devastating for vascular homeostasis. The direct effects of Cr(VI) in mediating cytotoxic effects have been linked to the ability of the metal to generate five-coordinated thiolato complexes with proteintyrosine phosphatase, which in a mechanism analogous with the isolelectronic vanadium(V) species, could inhibit phosphatase activity (36). Because phosphorylation is an important regulatory mechanism of eNOS activity, it is likely that Cr(VI)-metabolites interfere with NOS activity by altering phosphorylation cycle in the cell. Reportedly, vanadium(IV)-mediated NOS inhibition has been linked to sustained eNOS-Thr495 phosphorylation causing acute pulmonary vasoconstriction (37). Whether or not Cr(VI) can mediate a similar mechanism remains to be clarified. In summary, NOS-stimulated Cr(VI) reduction may be an important mechanism in the cytotoxic and vascular effects of pollution. It is likely that NOS will support a cytotoxic mechanism involving Cr(V)/Cr(IV) balance as previously suggested.

Chromium(V) Generation by NOS

Acknowledgment. We thank Dr. Bettie Sue Masters for critical reading of the manuscript. This work was supported in part by National Institutes of Health grant HL67244 to J.V.V. M.J. and P.M. were supported in this work by NIH grants HL30050 and GM52419 and a grant from The Robert A. Welch Foundation (AQ-1192) to Dr. Bettie Sue Masters. P.M. was also supported in part by MSMT, Czech Republic (grant LN 00A079).

References (1) Petronella, S. A., and Conboy-Ellis, K. (2003) Asthma epidemiology: risk factors, case finding, and the role of asthma coalitions. Nurs. Clin. North Am. 38, 725-735. (2) Gershwin, L. J. (2003) Effects of air pollutants on development of allergic immune responses in the respiratory tract. Clin. Dev. Immunol. 10, 119-126. (3) Sunyer, J. (2001) Urban air pollution and chronic obstructive pulmonary disease: a review. Eur. Respir. J. 17, 1024-1033. (4) Dockery, D. W., Pope, C. A., Xu, X., Spengler, J. D., Ware, J. H., Fay, M. E., Ferris, B. G., and Speizer, F. E. (1993) An association between air pollution and mortality in six US cities. N. Engl. J. Med. 329, 1753-1759. (5) Brook, R. D., Brook, J. R., Urch, B., Vincent, R., Rajagopalan, S., and Silverman, F. (1992) Inhalation of fine particulate air pollution and ozone causes acute arterial vasoconstriction in healthy adults. Circulation 105, 1534-1536. (6) Peters, A., Dockery, D. W., Muller, J. E., and Mittleman, M. A. (2001) Increased particulate air pollution and the triggering of myocardial infarction. Circulation 103, 2810-2815. (7) Magari, S. R., Hauser, R., Schwartz, J., Williams, P. L., Smith, T. J., and Christiani, D. C. (2001) Association of heart rate variability with occupational and environmental exposure to particulate air pollution. Circulation 104, 986-991. (8) Maheswaran, R., and Elliott, P. (2003) Stroke mortality associated with living near main roads in England and Wales: a geographical study. Stroke 34, 2776-2780. (9) Lukaski, H. C. (1999) Chromium as a supplement. Annu. Rev. Nutr. 19, 279-302. (10) Board on Agriculture, Committee on Animal Nutrition, Subcommittee on Mineral Toxicity in Animals. (1980) Chromium, in Mineral Tolerance of Domestic Animals, pp 142-163, National Academy Press, Washington D.C. (11) Suwa, T., Hogg, J. C. Quinlan, K. B., Ohgami, A., Vincent, R., and van Eeden, S. F. (2002) Particulate air pollution induces progression of atherosclerosis. J. Am. Coll. Cardiol. 39, 935-942. (12) Cuzzocrea, S., Persichini, T., Dugo, L., Colasanti, M., and Musci, G. (2003) Copper induces type II nitric oxide synthase in vivo. Free Radical Biol. Med. 34, 1253-1262. (13) Zhang, W. J., and Frei, B. (2003) Intracellular metal ion chelators inhibit TNF alpha-induced SP-1 activation and adhesion molecule expression in human aortic endothelial cells. Free Radical Biol. Med. 34, 674-682. (14) Pritchard, K. A., Jr., Ackerman, A., and Kalyanaraman, B. (2000) Chromium (VI) increases endothelial cell expression of ICAM-1 and decreases nitric oxide activity. J. Environ. Pathol. Toxicol. Oncol. 19, 251-260. (15) van Eeden, S. F., Tan, W. C., Suwa, T., Mukae, H., Terashima, T., Fuji, T., Qui, D., Vincent, R., and Hogg, J. C. (2001) Cytokines involved in the systemic inflammatory response induced by exposure to particulate matter air pollutants (PM(10)). Am. J. Respir. Crit. Care 164, 826-830. (16) Peters, A., Frohlich, M., Doring, A., Immervoll, T., Wischman, H. E. Hutchinson, W. L., Pepys, M. B., and Koenig, W. (2001) Particulate air pollution is associated with an acute phase response in men; results from the MONICA-Augsburg study. Eur. Heart J. 22, 1198-1204. (17) Bagchi, D., Stohs, S. J., Downs, B. W., Bagchi, M., and Preuss, H. G. (2002) Cytotoxicity and oxidative mechanisms of different forms of chromium. Toxicology 180, 5-22. (18) Zhao, H., Joseph, J., Zhang, H., Karoui, H., and Kalyanaraman, B. (2001) Synthesis and biochemical applications of a solid cyclic nitrone spin trap: a relatively superior trap for detecting superoxide anions and glutathiyl radicals. Free Radical Biol. Med. 31, 599-606.

Chem. Res. Toxicol., Vol. 18, No. 5, 2005 843 (19) Marta´sek, P., Liu, Q., Liu, J., Roman, L. J., Gross, S. S., Sessa, W. C., and Masters, B. S. (1996) Characterization of bovine endothelial nitric oxide synthase expressed in E. coli. Biochem. Biophys. Res. Commun. 219, 359-365. (20) Miller, T. R., Marta´sek, P., Roman, L. J., Nishimura, J. S., and Masters, B. S. S. (1997) Involvement of the reductase domain of neuronal nitric oxide synthase in superoxide anion production. Biochemistry 36, 15277-15284. (21) Roman, L. J., Marta´sek, P., Miller, R. T., Harris, D. E., de la Gaza, M. A., Shea, T. M., Kim, J.-J. P., and Masters, B. S. S. (2000) The C-termini of constitutive nitric-oxide synthases control electron flow through the flavins and heme domains and affect modulation by calmodulin. J. Biol. Chem. 275, 29225-29232. (22) Stearns, D., and Wetterhahn, K. E. (1997) Intermediates produced in the reaction of chromium (VI) with dehydroascorbate cause single-strand breaks in plasmid DNA. Chem. Res. Toxicol. 10, 271-278. (23) Roman, L. J., McLain, J., and Masters, B. S. (2003) Chimeric enzymes of cytochrome P450 oxidoreductase and neuronal nitricoxide synthase reductase domain reveal structural and functional differences. J. Biol. Chem. 278, 25700-25707. (24) Headlam, H. A., Weeks, C. L., Turner, P., Hambley, T. W., and Lay, P. A. (2001) Dinuclear chromium (V) amino acid complexes from the reduction of chromium (V) amino acid complex. Inorg. Chem. 40, 5097-5105. (25) Nishida, C. R., and Ortiz de Montellano, P. R. (2001) Control of electron transfer in nitric-oxide synthases. J. Biol. Chem. 276, 20116-20124. (26) Va´squez-Vivar, J., Hogg, N., Marta´sek, P., Karoui, H., Pritchard, K. A., Jr., and Kalyanaraman, B. (1999) Tetrahydrobiopterindependent inhibition of superoxide generation from neuronal nitric oxide synthase. J. Biol. Chem. 274, 26736-2642. (27) Perry, J. M., and Marletta, M. A. (1998) Effects of transition metals on nitric oxide synthase catalysis. Proc. Natl. Acad. Sci. U.S.A. 95, 11101-11106. (28) Persechini, A., McMillan, K., and Masters, B. S. S. (1995) Inhibition of nitric oxide synthase activity by Zn2+ ion. Biochemistry 34, 15091-15095. (29) Shi, X., and Dalal, N. S. (1994) Generation of hydroxyl radical by chromate in biologically relevant sytems: Role of Cr(V) complexes versus tetraperoxochromate(V). Environ. Health Perspect. 102, 231-236. (30) Codd, R., and Lay, P. A. (2003) Oxochromium(V) species formed with 2,3-dehydro-2-deoxy-N-acetylneuraminic (sialic) acids: An in vitro model system of oxochromium(V) species potentially stabilized in the respiratory tract upon inhalation of carcinogenic chromium (VI) compounds. Chem. Res. Toxicol. 16, 881-892. (31) Headlam, H. A., and Lay, P. A. (2001) EPR spectroscopic studies of the reduction of chromium(VI) by methanol in the presence of peptides. Formation of long-lived chromium(V) peptide complexes. Inorg. Chem. 40, 78-86. (32) Cotton, F. A., Dalal, N. S., Hillard, E. A., Huang, P., Murillo, C. A., and Ramsey, C. M. (2003) Applications of high field (W-Band) EPR to M-M bonded units (M ) Cr, Mo): The first confirmed oxidation of a Cr24+ paddlewheel complex to a satable isostructural Cr25+ product. Inorg. Chem. 45, 1388-1390. (33) Leonard, S., Wang, S., Zang, L., Castranova, V., Vallyathan, V., and Shi, X. (2000) Role of molecular oxygen in the generation of hydroxyl and superoxide anion radicals during enzymatic Cr(VI) reduction and its implication to Cr(VI)-induced carcinogenesis. J. Environ. Pathol. Toxicol. Oncol. 19, 49-60. (34) Levina, A., Barr-David, G., Codd, R., Lay, P. A., Dixon, N. E., Hammershoi, A., and Hendry, P. (1999) In vitro plasmid DNA cleavage by chromium (V) and -(IV) 2-hydroxcarboxylato complexes. Chem. Res. Toxicol. 12, 371-381. (35) Gaggeli, E., Berti, F., D’Amelio, N., Gagelli, N., Valensin, G., Bovalini, L., Paffetti, A., and Trabalzini, L. (2002) Metabolic pathways of carcinogen chromium. Env. Health Perspec. 110, 733-738. (36) Levina, A., and Lay, P. A. (2004) Solution structures of chromium (VI) complexes with glutathione and model thiols. Inorg. Chem. 43, 324-335. (37) Li, Z., Carter, J. D., Dailey, L. A., and Huang, Y.-C. T. (2004) Vanadyl sulfate inhibits NO production via threonine phosphorylation of eNOS. Environ. Health Perspect. 112, 201-206.

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