eNOS activation and NO function: structural motifs responsible for the posttranslational control of endothelial nitric oxide synthase activity

Abstract

Rather than being a constitutive enzyme as was first suggested, endothelial nitric oxide synthase (eNOS) is dynamically regulated at the transcriptional, posttranscriptional, and posttranslational levels. This review will focus on how changes in eNOS function are conferred by various posttranslational modifications. The latest knowledge regarding eNOS targeting to the plasma membrane will be discussed as the role of protein phosphorylation as a modulator of catalytic activity. Furthermore, new data are presented that provide novel insights into how disruption of the eNOS dimer prevents eNOS uncoupling and the production of superoxide under conditions of elevated oxidative stress and identifies a novel regulatory region we have termed the 'flexible arm'.

Figure 1

Homology model mediated reconstruction of the flexible arm region of human eNOS. Cysteines 94 and 99 from adjacent subunits form a zinc-tetrathiolate cluster (ZnS₄) that stabilizes the eNOS dimer. The ZnS₄ cluster appears to maintain the N-terminal region in a rigid conformation. It is also apparent that the catalytic center of eNOS is separated from the ZnS₄ cluster and no direct contacts are likely between them (A). Utilizing the Yasara modeling software, the ‘flexible arm’ region of human eNOS (corresponding to amino acids 105–125) was reconstructed. The ‘flexible arm’ connects the ZnS₄ cluster with the rest of the molecule (A). Also shown are the relative locations of the active site cavity, the ZnS₄ cluster, and the BH₄ binding site to the ‘flexible arm’ region (A). The ‘flexible arm’ appears to be seated under the cavity in the dimeric conformation with an intact ZnS₄ cluster. Molecular dynamic simulations predict that disruption of the ZnS₄ cluster will result in closure of the entrance to the catalytic cavity (arrow) of eNOS (B). Zoomed region of the structures with molecular surface are shown representing human eNOS with an intact (left) and disrupted ZnS₄ cluster (right) after molecular dynamic simulation (B). Flexible arm appeared in the substrate channel (blue) and blocked access to the heme after Zn removal.

Figure 2

Flexible arm feature of eNOS compared to bacterial NOS (bsNOS) and cytochrome P450. Panel A demonstrates the structural similarity of bsNOS (magenta) and eNOS (yellow). The N-terminal end of bsNOS begins immediately after the flexible arm region (red) of eNOS. Thus, bsNOS is deficient in both the redox regulation. Panel B demonstrates the superposition of open (yellow) and closed (magenta) conformations of P450. This flexible region of P450 controls substrate access to the heme and thus serves the same function as the eNOS flexible arm. However, as the P450 lacks the ZnS₄ cluster, it is not susceptible to redox regulation.

Figure 3

The effect on NO signaling in endothelial cells challenged by H₂O₂. The response of EC with regard to NO generation in response to oxidative stress can be divided into two pathways: a physiologic response (right) and a pathologic response (left). The physiologic/pathologic decision is then predicated on the level of H₂O₂ to which the cell is exposed, the duration of the stimulus, and the ability of the cell to metabolize it.

Figure 4

Human eNOS ZnS₄ cluster mutants have reduced dimer levels. The dimer levels of human eNOS ZnS₄ cluster mutants purified as described (Ravi et al. 2004) was assessed by analytical gel filtration (A), again as described (Ravi et al. 2004). Analysis indicates that the dimer (D) levels are decreased and monomer (M) levels are increased in ZnS₄ mutants (A). Images are representative of n=3. The absorbance spectra of wild-type and ZnS₄ mutants were also analyzed (B). The Soret absorption maximum in wild-type eNOS is found at 395 nm and corresponds to the heme bound to L-arginine and BH₄ (B). A red shift in the Soret band maximum to 412 nm is observed for the ZnS₄ mutants corresponding to L-arginine and BH₄ dissociation (B). The data presented are the mean from three independent experiments. Shown in the insert is a scheme depicting the metal to ligand charge transfer (MLCT) and ‘Soret band’ interference (B).

Figure 5

Human eNOS ZnS₄ cluster mutants have reduced heme accessibility for cyanide. U.V.–visible spectra of the cyanide–iron complex were measured on a Shimadzu spectrophotometer in the region from 350 to 600 nm. Recombinant wild-type (A) and the ZnS₄ cluster mutants of eNOS (B–D) were dissolved in PBS to final concentration 0·1 mg/ml. The initial spectrum was obtained before addition of KCN and then the spectrum was recorded after each addition of 1 μl KCN solution (125 mM stock). The addition of 1 μl KCN stock solution was repeated until the reaction reached saturation, as determined by no further changes in absorption at 412 nm. There is a dose-dependent increase in KCN binding to the wild-type protein (A) that does not occur in any of the ZnS₄ cluster mutants (B–D). The data presented are the mean±S.D. from three independent experiments.

Figure 6

Human eNOS ZnS₄ cluster mutants have reduced heme accessibility for imidazole. U.V.–visible spectra of the imidazole–iron complex were measured on a Shimadzu spectrophotometer in the region from 350 to 600 nm. Recombinant wild-type (A) and the ZnS₄ cluster mutants of eNOS (B–D) were dissolved in PBS to final concentration 0·1 mg/ml. The initial spectrum was obtained before addition of imidazole and then the spectrum was recorded after each addition of 1 μl imidazole solution (100 mM stock). The addition of 1 μl imidazole stock solution was repeated until the reaction reached saturation, as determined by no further changes in absorption at 430 nm. There is a dose-dependent increase in imidazole binding to the wild-type protein (A) that does not occur in any of the ZnS₄ cluster mutants (B–D). The data presented are the mean±S.D. from three independent experiments.

Figure 7

Endothelial NOS mutants with altered heme and BH₄ binding do not exhibit altered heme accessibility. Recombinant mutants of eNOS with altered heme (C184A) and BH₄ binding (W445A) were exposed to increasing concentrations of potassium cyanide (KCN, 0–12 mM) (A and B) or imidazole (C and D) as described in Fig. 6 and the changes in absorbance between 350 and 600 nm determined. There is a dose-dependent increase in both KCN and imidazole binding in the C184A- and W445A-eNOS mutant proteins. The data presented are the mean±S.D. from three independent experiments.

Figure 8

NO and superoxide production by wild-type and the ZnS₄ cluster mutants of eNOS. Recombinant wild-type eNOS and the ZnS₄ cluster mutants (C94A, C99A, and C9499A) were incubated with L-arginine and the appropriate co-factors for 30 min at 37 °C and NO (A) and superoxide levels (B) determined as described (Grobe et al. 2006). There is a significant reduction in both NO (A) and superoxide (B) generation in the ZnS₄ cluster mutants. Data are presented as mean±S.E.M.; n=3. *P<0·05 versus wild-type eNOS. ^†P<0·05 versus C94A mutant.

Figure 9

Molecular dynamic simulations identify the amino acids in the flexible arm of human eNOS responsible for closure of the heme cavity. Superposition of intact eNOS (green) and with disrupted ZnS₄ (yellow) represents the flexible arm movement. Molecular dynamic simulations predict conformational changes in the flexible arm region that are driven by salt bridge formation between positively charged amino acids (blue) of the flexible arm (Arg107, Lys108, and Arg112) and negatively charged amino acids on the wall of the substrate channel.

Figure 10

The human eNOS flexible arm mutant undergoes self-nitration. Purified wild-type recombinant eNOS was compared to the flexible arm mutant contained on a disrupted ZnS₄ cluster background (C94A/C99A/R107A/K108A/R112A). The flexible arm mutant has dimer (D) and monomer (M) levels, as determined by analytic gel filtration, that are similar to those seen with wild-type eNOS (wt eNOS, A). The absorbance spectrum of the flexible arm mutant contains a Soret band at 395 nm that is comparable with wild-type eNOS (B). The flexible arm mutation also allows both cyanide (C) and imidazole (D) to access the heme. The flexible arm mutant produces NO at levels comparable to the wild-type protein (E). However, the flexible arm mutant significantly generates greater levels of both superoxide (F) and ONOO⁻ (G) compared with the wild-type protein. Western blot analysis using an antibody specific for 3-NT residues indicates that the flexible arm mutant undergoes increased self-nitration compared with either wild-type eNOS or the ZnS₄ cluster mutants (C94A, C99A, and C94A/99A, H). A representative image is shown. Data are presented as mean±S.E.M.; n=3. *P<0·05 versus wild-type eNOS.