Inhibition of nitric oxide synthase by cobalamins and cobinamides

Abstract

Cobalamins are important cofactors for methionine synthase and methylmalonyl-CoA mutase. Certain corrins also bind nitric oxide (NO), quenching its bioactivity. To determine if corrins would inhibit NO synthase (NOS), we measured their effects on -L-[(14)C]arginine-to-L-[(14)C]citrulline conversion by NOS1, NOS2, and NOS3. Hydroxocobalamin (OH-Cbl), cobinamide, and dicyanocobinamide (CN(2)-Cbi) potently inhibited all isoforms, whereas cyanocobalamin, methylcobalamin, and adenosylcobalamin had much less effect. OH-Cbl and CN(2)-Cbi prevented binding of the oxygen analog carbon monoxide (CO) to the reduced NOS1 and NOS2 heme active site. CN(2)-Cbi did not react directly with NO or CO. Spectral perturbation analysis showed that CN(2)-Cbi interacted directly with the purified NOS1 oxygenase domain. NOS inhibition by corrins was rapid and not reversed by dialysis with L-arginine or tetrahydrobiopterin. Molecular modeling indicated that corrins could access the unusually large heme- and substrate-binding pocket of NOS. Best fits were obtained in the "base-off" conformation of the lower axial dimethylbenzimidazole ligand. CN(2)-Cbi inhibited interferon-gamma-activated Raw264.7 mouse macrophage NO production. We show for the first time that certain corrins directly inhibit NOS, suggesting that these agents (or their derivatives) may have pharmacological utility. Endogenous cobalamins and cobinamides might play important roles in regulating NOS activity under normal and pathological conditions.

Figures

Figure 1. General structure of corrins/cobalamins

The figure demonstrates various upper axial ligands, the corrin ring, and a lower axial ligand. Hydroxocobalamin has a hydroxyl group at the R position. Cyanocobalamin has a cyano group at the R position. Methylcobalamin and adenosylcobalamin have a methyl and adenosyl group, respectively, at the R position. Cobinamide lacks the lower axial ligand and has a hydroxyl group at the R position. Dicyanocobinamide has a cyano group as the lower axial ligand and at the R position.

Figure 2. A–D. Inhibition of NOS1, NOS2, and NOS3 enzymatic by cobinamides and cobalamins

The enzymatic activity of purified NOS1, NOS2, or NOS3 was assessed in presence or absence of different concentrations of (A) hydroxocobalamin, (B) cobinamide, (C) dicyanocobinamide, or (D) cyanocobalamin. Results are expressed as percent inhibition. The estimated effective dose for 50% inhibition (ED50) is displayed. Note the differing scales of the x axes.

Figure 3. A–D. Influence of light on inhibition of purified, recombinant human NOS1 and NOS2 by methylcobalamin and adenosylcobalamin

The effect of methylcobalamin (not exposed to light or exposed to light) and adenosylcobalamin (not exposed to light or exposed to light) on NOS1 (A and C) or NOS2 (B and D) activity is displayed. The agents were protected from light or exposed to light for 60 minutes. We performed the enzyme assays in near-dark conditions (as low light as possible). Results are expressed as percent inhibition. The effective dose for 50% inhibition (ED50) is displayed. Light dissociates the methyl- or adenosyl- group from the agent resulting in hydroxocobalamin. Methylcobalamin and adenosylcobalamin have very little NOS inhibitory activity, but after light-induced change to hydroxocobalamin, they actively inhibit NOS1 and NOS2 activity.

Figure 4. Effect of dicyanocobinamide on the UV-visible spectra of purified, recombinant NOS1 oxygenase domain

Binding of 40 μM CN2Cbi to NOS1 oxygenase domain (NOS1ox; ~ 10 μM) in 40 mM bis- tris-propane buffer (pH 7.5) containing 5% glycerol and 10 μM H4B with at room temperature. The solid line represents NOS1ox alone, and the dashed line NOS1ox with CN2Cbi after 200 minutes’ interaction. The inset displays the difference spectrum generated by subtracting NOS1ox initial spectrum from the spectrum after 200 minutes with CN2Cbi.

Figure 5. A–D. Spectroscopy of dicyanocobinamide interactions with purified, recombinant NOS2 oxygenase domain, or NO

Dicyanocobinamide reacts directly with purified, recombinant NOS2 oxygenase domain (A & B): Oxygen reacts directly with the reduced iron of the heme group in NOS. We used carbon monoxide (CO) (that simulates O2 in binding to the reduced iron) to determine the influence of CN2-Cbi on oxygen binding to NOS. The reaction of purified NOS2 oxygenase domain (approximately 3 μM final concentration) with CO in a reduced state (with dithionite) markedly changed the UV/vis spectrum, with appearance of the typical CO-bound Fe+2-heme peak at 445 nm (A). However, in the presence of CN2-Cbi, there was no appearance of the 445 nm peak induced by CO in a reduced state using NOS2 oxygenase domain at a final concentration of approximately 4.5 μM (B). Similar findings were noted with OH-Cbl instead of CN2-Cbi. Hydroxocobalamin binds NO, but dicyanocobinamide does not (C & D). NO reacts with OH-Cbl (C) as indicated by the change in the UV/Vis spectrum (shift of peak from 352 nm to 354–355 nm, and alteration in the 520 nm region). However, NO does not react with CN2-Cbi (D) (no change in spectrum).

Figure 6. Structural studies of cobalamin-NOS interactions

6A. Structures of adenosylcobalamin in the “base-on” and “base-off” configurations showing the positions of the corrin ring and the dimethylbenzimidazole moiety. The purple spheres represent the Van der Waals surface of the cobalt atom. The structure of the “base-off” form was taken from the structure of glutamate mutase reported by Reitzer et al (PDB entry 1cb7) [44]. In this structure, the cobalt ligand trans to the methyl axial ligand is a histidyl residue (not shown) supplied by the protein. The “base-on” form shown was taken from the structure of cobalamin bound to transcobalamin reported by Wuerges et al (PDB entry 2bb5) [45]. The axial ligand trans to dimethylbenzimidazole is again a histidyl residue (not shown) supplied by the protein. Slight differences in the planarity of the corrins include movement of the cobalt towards dimethylbenzimidazole in the “base-on” configuration. Considerable flexibility in the molecule exists, making many other orientations of the base possible. 6B. Docking of the “base-off” form of cobalamin (as in Figure 5A) in the heme pockets of NOS2 (dark blue) and NOS1 (cyan), illustrating the extent of the ligand-binding cavity relative to the size of cobalamin. Heme is shown in red and cobalamin in purple. The very large substrate-binding pocket accommodates surprisingly large ligands, including porphyrins (similar in size and shape to the corrin shown here). The insertion of cobalamin into the heme pocket is ultimately limited by a beta sheet near V567 (NOS1). The configuration shown generates a 0.4Angstrom clash with the valine side chain in the crystal structure from PDB P_1ZVL, and represents the approximate limit of insertion of cobalamin, assuming slight rearrangements in the backbone are possible. A similar configuration with approximately 80% overlap of the corrin and heme can be obtained without backbone or unrelaxable side chain clashes. Slight torsioning of some active site residue side chains and repositioning of the DMBzI moiety of the cobalamin (visible as rings in solid rendering at the upper right) was necessary. The aromatic side chains visible at the bottom of the figure at the edge of the access channel are W306 and Y706 in NOS1.

Figure 7. Dicyanocobinamide inhibition of NO formation by mouse macrophages

Cells of the mouse macrophage cell line Raw 264.7 were incubated 24 hours with 500 units/ml murine interferon gamma to activate them for NO production. The cultures also contained 0, 0.5, 2.0, or 8.0 μM dicyanocobinamide for the entire culture period. At the end of the culture period, the cells were assessed for NO production using DAF-FM and a fluorimeter. Results are expressed as NO production (percent of control). The error bars show the SEM. This is from 6 separate experiments.