Abstract
Electron paramagnetic resonance was used to characterize the heme structure of resting endothelial nitric-oxide synthase (eNOS), eNOS devoid of its myristoylation site (G2A mutant), and their heme complexes formed with 16 different ligands. Resting eNOS and the G2A mutant have a mixture of low spin and high spin P450-heme with widely different relaxation behavior and a stable flavin semiquinone radical identified by EPR as a neutral radical. This flavin radical showed efficient electron spin relaxation as a consequence of dipolar interaction with the heme center; P1/2 is independent of Ca2+-calmodulin and tetrahydrobiopterin. Seven of the 16 ligands led to the formation of low spin heme complexes. In order of increasing rhombicity they are pyrimidine, pyridine, thiazole, L-lysine, cyanide, imidazole, and 4-methylimidazole. These seven low spin eNOS complexes fell in a region between the P and O zones on the "truth diagram" originally derived by Blumberg and Peisach (Blumberg, W. E., and Peisach, J. (1971) in Probes and Structure and Function of Macromolecules and Membranes (Chance, B., Yonetani, T., and Mildvan, A. S., eds) Vol. 2, pp. 215-229, Academic Press, New York) and had significant overlap with complexes of chloroperoxidase. A re-definition of the P and O zones is proposed. As eNOS and chloroperoxidase lie closer than do eNOS and P450cam on the truth diagram, it implies that the distal heme environment in eNOS resembles chloroperoxidase more than P450cam. In contrast, 4-ethylpyridine, 4-methylpyrimidine, acetylguanidine, ethylguanidine, 2-aminothiazole, 2amino-4,5-dimethylthiazole, L-histidine, and 7-nitroindazole resulted in high spin heme complexes of eNOS, similar to that observed with L-arginine. This contrasting EPR behavior caused by families of ligands such as imidazole/L-histidine or thiazole/2-aminothiazole confirms the conclusion derived from parallel optical and kinetic studies. The ligands resulting in the low spin complexes bind directly to the heme iron, while their cognate ligands induce the formation of high spin complexes by indirectly perturbing the heme structure and excluding the original axial heme ligand in the resting eNOS (V. Berka, P.-F. Chen, and A. -L. Tsai (1997) J. Biol. Chem. 272, in press). The difference in EPR spectra of these high spin eNOS complexes, although subtle, are different for different homologs.
Highlights
The free radical nitric-oxide serves as an important messenger in a wide variety of physiological and pathophysiological processes
electron paramagnetic resonance (EPR) and Heme Spin State of the Wild-type endothelial NOS (eNOS) and G2A Mutant—Wild-type eNOS isolated from insect cells appears to contain both high and low spin heme as revealed by its EPR spectrum (Fig. 1, top panel) and as was reflected in the location of its Soret maximum of 398 – 400 nm2 [25]
There are two low spin heme complexes resolved in the EPR measured at 0.2 mW (Fig. 3, spectrum a), each with distinctive g values of 2.54, 2.29, 1.84 and 2.42, 2.29, 1.90, respectively (Table I)
Summary
2-methylimidazole, 4-methylimidazole, pyridine, 4-ethylpyridine, pyrimidine and 4-methylpyrimidine, thiazole, 2-aminothia-. Recombinant human endothelial nitric-oxide synthase (eNOS) was prepared using a baculovirus expression system as described previously with slight modification [15, 16]. Soluble endothelial nitric-oxide synthase was prepared by specific replacement of the second amino acid, glycine, to alanine through oligonucleotide-directed mutagenesis described elsewhere.. Activity of eNOS was measured as the conversion of tritium-labeled L-arginine to L-citrulline as described previously [15, 16]. In the case of low spin heme, the gmin (gx) was either measured directly, or calculated from [20, 21]. For the high spin heme, gx and gy were measured directly, and E and D were calculated using Equation 5. Gz was measured directly when observable, otherwise it was calculated using Equation 6 and the previously determined values of E and D.
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