Jianmongkol S, Vuletich JL, Bender AT, Demady DR, Osawa Y. 2000. Aminoguanidine-mediated inactivation and alteration of neuronal nitric oxide synthase. J Biol Chem 275(18):13370–13376.
It is established that aminoguanidine (AG) is a metabolism-based inactivator of the three major isoforms of nitric-oxide synthase. AG is thought to be of potential use in diseases, such as diabetes, where pathological overproduction of NO is implicated. We show here that during the inactivation of neuronal nitric-oxide synthase (nNOS) by AG that the prosthetic heme is altered, in part, to dissociable and protein-bound adducts. The protein-bound heme adduct is the result of cross-linking of the heme to residues in the oxygenase domain of nNOS. The dissociable heme product is unstable and reverts back to heme upon isolation. The alteration of the heme is concomitant with the loss in the ability to form the ferrous-CO complex of nNOS and accounts for at least two-thirds of the activity loss. Studies with [14C]AG indicate that alteration of the protein, in part on the reductase domain of nNOS, also occurs but at low levels. Thus, heme alteration appears to be the major cause of nNOS inactivation. The elucidation of the mechanism of inactivation of nNOS will likely lead to a better understanding of the in vivo effects of NOS inhibitors such as AG.
Nitric-oxide synthases (NOS)1 are cytochrome P-450-like hemoprotein enzymes that catalyze the conversion ofL-arginine to citrulline and nitric oxide by a process that requires NADPH and molecular oxygen (1-4). NOSs are bidomain in structure with an oxygenase domain, which contains the cysteine residue that is coordinated to the prosthetic heme as well as the tetrahydrobiopterin binding site, and a reductase domain, which contains the binding sites for FMN, FAD, and NADPH (5). For the neuronal and endothelial isoforms, the activity of the enzyme is regulated by calcium and calmodulin, which binds to a site between the oxygenase and reductase domains. In the case of the macrophage or inducible NOS (iNOS), the calmodulin is bound very tightly and is not regulated by physiological levels of calcium (6).
The isoform-selective inhibition of NOS has been of intense interest because of the various physiological and pathological actions of nitric oxide caused by the three isoforms (for reviews see Refs. 7-11). Our laboratory has focused on the inhibition of neuronal NOS (nNOS) by guanidine drugs (12). Aminoguanidine (AG) is structurally one of the simplest of these drugs containing a guanidino moiety and a hydrazine group. AG is thought to be of potential use in diabetes (11, 13), and it was first reported by Corbett et al. (14) to inhibit iNOS in a selective manner. The selectivity for iNOS has been confirmed in various models (11, 15), although in some studies the selectivity appears to be limited (16, 17). More recently, it was shown that AG is an isoform-selective mechanism-based inactivator of iNOS in vitro, although the constitutive NOS in GH3 pituitary cells and that in endothelial cells could also be inactivated (18). Diaminoguanidine, which is structurally related to AG, is also a mechanism-based inactivator of all three isoforms of NOS, although it exhibits less selectivity for iNOS (19). The mechanism by which AG causes the inactivation of NOS is not known. In studies with the use of [14C]AG, it has been shown that inactivation of iNOS causes radiolabel to be associated in part with iNOS protein (20). In the same study, the fluorescence caused by the heme was not altered by AG treatment of iNOS, indicating that the prosthetic heme was not destroyed. HPLC analysis of iNOS treated with [14C]AG showed radiolabel associated with a peak that eluted with “near identity” to heme, and the authors suggest that this was an altered heme product, but this product was not further characterized (20). Moreover, it is not clear how much of the prosthetic heme is altered in relation to iNOS inactivation from these studies. More recently, similar to the findings on iNOS, the inactivation of nNOS by AG has been found to occur without changes in the heme fluorescence and to lead to irreversible binding of [14C]AG to the protein (21). Thus, it appears that AG can alter the heme or protein of NOS, but the role for either of these processes in the inactivation has not been demonstrated
In the current study, we have sought to define the mechanism by which AG inactivates recombinant nNOS. We demonstrated that AG causes the alteration of the prosthetic heme in part to a dissociable adduct as well as to a protein-bound adduct, which was primarily the result of heme binding irreversibly to a site(s) on the oxygenase domain. The alteration of the heme was concomitant to the loss in the ability to form the ferrous-CO complex and appears to be the major cause for the loss of enzyme activity. Inactivation of nNOS with [14C]AG gave radiolabel associated with the dissociable heme product as well as radiolabel irreversibly bound to the protein primarily at a site(s) on the reductase domain of nNOS. These studies further our understanding of the mechanisms involved in the inactivation of nNOS and may aid in predicting the action of NOS inhibitors in vivo.