Characterization of the roles of the 594-645 region in human endothelial nitric-oxide synthase in regulating calmodulin binding and electron transfer

Abstract

It has been postulated that a segment (residues 594645) inserted in the FMN subdomain of human endothelial nitric-oxide synthase (eNOS) plays a crucial role in controlling Ca2+-dependent CaM binding for eNOS activity. To investigate its functions, we expressed human eNOS in a baculovirus system with deletion of a 45-residue segment from this region (residues 594-606 and 614-645, designated as Δ45eNOS), and characterized the purified mutant enzyme. In contrast with wild-type eNOS, Δ45eNOS exhibited characteristics resembling inducible NOS (iNOS). It contained an endogenously bound CaM, which was essential in folding and stabilizing this mutant enzyme, and retained 60% of L-citrulline formation in 5 mM EGTA. We also produced four N- terminally truncated reductase domains with or without the 45-residue segment, and either including or excluding the CaM-binding sequence. Basal cytochrome c reductase activity of reductase domains without the 45-residue segment was up to 20 fold greater than that of corresponding insert- containing domains, and higher than CaM-stimulated activity of the wild-type enzyme. A series of mutants with smaller fragment deletion in this region such as Δ594-604, Δ605-612, Δ613-625, Δ626-634, Δ632-639, and Δ640-645 mutants were further characterized. The crude lysate of mutants Δ613-625 and Δ632-639 did not show activity in the presence of Ca2+/CaM, while other four mutants had activity comparable to that of WTeNOS. The purified Δ594- 604 and Δ605-612 proteins had a 3-5-fold higher affinity for Ca2+/CaM, but their L-citrulline forming activity was still 80% dependent upon the addition of Ca2+/CaM. Both mutants exhibited a low level of the cytochrome c and ferricyanide reductase activities, which either did not respond to (Δ594- 604) or slightly enhanced by (Δ605-612) the exogenous CaM. In contrast, activities of Δ626-634 and Δ640-645 like those of WTeNOS were largely Ca2+/CaM-dependent. Thus, our findings indicate that the N-terminal half of the 594-645 segment containing residues 594-612 plays a significant role in regulating Ca2+/CaM binding.