Electron Transfer and Catalytic Activity of Nitric Oxide Synthases

Abstract

The nitric oxide synthases (NOS) are single polypeptides that encode a heme domain, a calmodulin binding motif, and a flavoprotein domain with sequence similarity to P450 reductase. Despite this basic structural similarity, the three major NOS isoforms differ significantly in their rates of ⅐NO synthesis, cytochrome c reduction, and NADPH utilization and in the Ca 2؉ dependence of these rates. To assign the origin of these differences to specific protein domains, we constructed chimeras in which the reductase domains of endothelial and inducible NOS, respectively, were replaced by the reductase domain of neuronal NOS. The results with the chimeric proteins confirm the modular organization of the NOS polypeptide chain and demonstrate that (a) similar residues establish the necessary contacts between the reductase and heme domains in the three NOS isoforms, (b) the maximal rate of ⅐NO synthesis is determined by the maximum intrinsic ability of the reductase domain to deliver electrons to the heme domain, (c) the Ca The flavin domain uncouples the electrons provided by NADPH and delivers them, one at a time, to the prosthetic heme iron atom. H 4 B is required for ⅐NO synthesis by all three proteins, but its role in the catalytic process remains unclear (9).Clear differences exist in the Ca 2ϩ and CaM dependence of the three NOS isoforms. The binding of CaM to nNOS and eNOS, the two constitutive isoforms, is Ca 2ϩ -dependent and reversible (10, 11), whereas the binding of CaM to iNOS is essentially a Ca 2ϩ -independent, irreversible process (12). Thus, the catalytic activities of nNOS and eNOS are regulated by cellular Ca 2ϩ -levels, whereas the activity of iNOS is insensitive to the Ca 2ϩ concentration and is primarily regulated by the rate at which the protein is synthesized (12). In addition to the heme, CaM, and reductase domains common to all the isoforms, nNOS has an additional N-terminal domain thought to be involved in subcellular targeting (13,14), and eNOS is unique in that it has myristoylation and palmitoylation sites that target it to the membrane (15, 16).The catalytic activities of the NOS isoforms differ considerably, regardless of whether activity is measured as ⅐NO formation or cytochrome c reduction. Regardless of which of these two parameters is measured, the activities of iNOS and nNOS are considerably higher than that of eNOS. Thus, in our hands, the V max values for ⅐NO formation by recombinant iNOS (17), nNOS (18), and eNOS (19) were found to be ϳ800, ϳ400, and ϳ130 nmol min Ϫ1 mg Ϫ1 , respectively, and the rates of electron transfer to cytochrome c in the presence of CaM and Ca 2ϩ were ϳ45,000, ϳ44,000, and ϳ1,800 nmol min Ϫ1 mg Ϫ1 , respectively. In each case, the reductase domain was able to transfer electrons to cytochrome c at a rate much greater than the maximum rate of ⅐NO production, although the nature of the ratelimiting step(s) in ⅐NO formation and the reason(s) for the differences in the catalytic activities of the isoforms are unclear. The reduction of cytochrome c by nNOS and eN...

Nitric-oxide Synthase (NOS) Reductase Domain Models Suggest a New Control Element in Endothelial NOS That Attenuates Calmodulin-dependent Activity

“…These differences in electron transfer activity with and without CaM can be observed when the nNOS heme and reductase domains are independently expressed (16,17). Furthermore, the elements that control the CaM dependence and relatively low rate of electron transfer to the heme domain in eNOS appear to be localized entirely within the reductase domain because NOS chimeras with the eNOS oxygenase domain fused to the iNOS or nNOS reductase domain exhibit high activities (18). Conversely, chimeras in which the eNOS reductase domain is fused to the iNOS or nNOS oxygenase domain have low, eNOS-like activities (18).…”

Electron Transfer, Oxygen Binding, and Nitric Oxide Feedback Inhibition in Endothelial Nitric-oxide Synthase

“…Conceivably, slow heme reduction in eNOS could involve structural elements in both its reductase and oxygenase domains. However, poor Fe(CN) 6 and cytochrome c reduction by the CaM-bound reductase domain (11), along with the fact that a NOS chimera comprised of an nNOS reductase and eNOS oxygenase domain had faster NO synthesis than native eNOS (30), suggest that structural elements responsible for slow heme reduction reside in the reductase domain. From our present work we conclude that slow heme reduction is not because of slow flavin reduction in eNOS, which is actually quite fast in the presence of CaM.…”

“…Because NO synthesis is actually the result of many steps, it is imperative to identify which steps limit the activity of a particular NOS isoform. Work with NOS chimeras containing swapped reductase domains has suggested heme reduction could be responsible for the low activity of eNOS (30). However, it seems that NOS catalysis is comprised of two parts (15,16): an active component that includes all steps involved in NO generation and an inactive component that includes NO binding to the NOS heme and subsequent dissociation or oxidation of the heme-NO complex to regenerate active enzyme.…”

Recruitment of governing elements for electron transfer in the nitric oxide synthase family

“…Because the proximal electron donor and acceptor are on the same polypeptide chain in the NOSs, and thus each potential acceptor is linked to its donor, unlike CYPOR and the cytochromes P450, regulation of electron transfer by the reductase domain is reasonable. In fact, the rate of electron transfer from the reductase to the heme domain determines the turnover rates of NO production in the various NOSs (30), and thus alterations, such as truncations or insertions in the reductase domain, might confer the ability to regulate electron transfer through this domain. The remarkable sequence identity (58%) between the C-terminal 641 amino acids of neuronal NOS and the entire sequence of CYPOR was reported by Bredt et al (31).…”