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. 1998 Aug 4;95(16):9220-5.
doi: 10.1073/pnas.95.16.9220.

Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors

J Vásquez-Vivar  1 , B Kalyanaraman, P Martásek, N Hogg, B S Masters, H Karoui, P Tordo, K A Pritchard Jr
Affiliations

Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors

J Vásquez-Vivar et al. Proc Natl Acad Sci U S A. .

Abstract

The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium/calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium/calmodulin binding, superoxide formation is increased. Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation. No inhibition is observed after addition of L-arginine, NG-hydroxy-L-arginine, L-thiocitrulline, and L-NG-monomethyl arginine to activated eNOS. These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous-dioxygen complex and that occupation of the L-arginine binding site does not inhibit this process. However, the concomitant addition of L-arginine and tetrahydrobiopterin (BH4) abolishes superoxide generation by eNOS. Under these conditions, L-citrulline production is close to maximal. Our data indicate that BH4 fully couples L-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous-dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of flavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.

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Figures

Figure 1
Figure 1
Calcium/calmodulin-dependent superoxide generation from eNOS. eNOS (7 pmol) was incubated with (A), 0.2 mM calcium, 20 μg/ml calmodulin, 0.1 mM NADPH, 50 mM DEPMPO, and 0.1 mM DTPA in 50 mM Hepes (pH 7.4). (B) As A, in the absence of Ca2+/calmodulin. (C) As A, in the presence of 10 μg/ml SOD. (D) As A, but without enzyme. Incubations were made at room temperature and the spectra were acquired by using a loop–gap resonator. Instrumental conditions: microwave power 2 mW, modulation amplitude 1 G, time constant 0.128 s, scan rate 1.6 G/s, gain 1.25 × 105, number of scans, 10. ESR data can be fitted by considering two isomers of DEPMPO-OOH (hyperfine coupling constants are given in Gauss). Isomer 1 (63% contribution), aN = 13.0, aP = 50.6, aH = 11.7; isomer 2 (37% contribution), aN = 13.1, aP = 48.4, aH = 9.9; and DEPMPO-OH (13% contribution), aN = 13.9, aP = 46.7, aH = 13.5.
Figure 2
Figure 2
Inhibition of eNOS-dependent superoxide generation by cytochrome P450 type inhibitors. eNOS (7 pmol) was incubated with 0.1 mM NADPH/50 mM DEPMPO in 50 mM Hepes (pH 7.4) containing 0.1 mM DTPA and (A) 0.2 mM calcium and 20 μg/ml calmodulin. (B) As A, in the presence of 0.5 mM imidazole. (C) As A, in the presence of 1 mM cyanide. (D) As A, after 15 min incubation with 0.1 mM phenyldiazene. ○, DEPMPO-OOH; •, DEPMPO-phenyl radical and was simulated (broken line in D) by using the hyperfine coupling constants (in Gauss). aN = 14.8, aP = 44.9, aH = 23.3. Instrumental conditions: microwave power 2 mW, modulation amplitude 1 G, time constant 0.128 s, scan rate 1.6 G/s, gain 1.25 × 105, number of scans, 10.
Figure 3
Figure 3
Effect of BH4 on the generation of superoxide by eNOS. eNOS (7 pmol) was incubated with (A) 0.2 mM calcium, 20 μg/ml calmodulin, 0.1 mM NADPH, 50 mM DEPMPO, and 0.1 mM DTPA in 50 mM Hepes (pH 7.4). (B) As A, in the presence of 1 μm BH4. (C) As A, in the presence of 10 μM BH4. (D) As A, in the presence of 100 μM BH4. (E) As C, in the presence of 0.1 mM l-arginine. Instrumental conditions: microwave power 2 mW, modulation amplitude 1 G, time constant 0.128 s, scan rate 1.6 G/s, gain 1.25 × 105, number of scans, 10.
Figure 4
Figure 4
Effects of l-arginine, BH4, and l-NAME on the generation of superoxide by eNOS. eNOS (7 pmol) was incubated with 0.2 mM calcium, 20 μg calmodulin, 0.1 mM NADPH, 50 mM DEPMPO, and 0.1 mM DTPA in 50 mM Hepes (pH 7.4) with (A) 1 μM FAD and 1 μM FMN. (B) As A, in the presence of 0.1 mM l-arginine. (C) As B, in the presence of 10 μM BH4. (D) As C, in the presence of 0.1 mM l-NAME. Instrumental conditions: microwave power 2 mW, modulation amplitude 1 G, time constant 0.128 s, scan rate 1.6 G/s, gain 1.25 × 105, number of scans, 10.
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