Distinct Influence of N-terminal Elements on Neuronal Nitric-oxide Synthase Structure and Catalysis

Abstract

Nitric oxide (NO) is a signal molecule produced in animals by three different NO synthases. Of these, only NOS I (neuronal nitric-oxide synthase; nNOS) is expressed as catalytically active N-terminally truncated forms that are missing either an N-terminal leader sequence required for protein-protein interactions or are missing the leader sequence plus three core structural motifs that in other NOS are required for dimer assembly and catalysis. To understand how the N-terminal elements impact nNOS structure-function, we generated, purified, and extensively characterized variants that were missing the N-terminal leader sequence (⌬296nNOS) or missing the leader sequence plus the three core motifs (⌬349nNOS). Eliminating the leader sequence had no impact on nNOS structure or catalysis. In contrast, additional removal of the core elements weakened but did not destroy the dimer interaction, slowed ferric heme reduction and reactivity of a hemedioxy intermediate, and caused a 10-fold poorer affinity toward substrate L-arginine. This created an nNOS variant with slower and less coupled NO synthesis that is predisposed to generate reactive oxygen species along with NO. Our findings help justify the existence of nNOS N-terminal splice variants and identify specific catalytic changes that create functional differences among them.

Surface Charges and Regulation of FMN to Heme Electron Transfer in Nitric-oxide Synthase

“…Full-length proteins were purified in the presence of H 4 B and L-Arg as described previously (23) but after the Ni 2ϩ nitrilotriacetic acid-Sepharose CL-4B column, 2 mM CaCl 2 was added to the sample and the 2Ј,5Ј-ADP-Sepharose column step was replaced by a CaM-Sepharose column as reported for the purification of the reductase domain (14). NOSoxy proteins were expressed and purified as described (24). Protein concentration was determined from the absorbance at 444 nm of the ferrous heme-CO complex, using an extinction coefficient of 76 mM Ϫ1 cm Ϫ1 (⌬⑀ 444 nm-500 nm ) (25).…”

Catalytic Reduction of a Tetrahydrobiopterin Radical within Nitric-oxide Synthase

“…Protein Purification-Rat nNOS was overexpressed in Escherichia coli and was purified in the absence of H 4 B as described previously (18,19). The enzyme concentration was determined from the 444-nm absorbance of the ferrous-CO complex using an extinction coefficient of 76 mM Ϫ1 cm Ϫ1 .…”

“…The enzyme concentration was determined from the 444-nm absorbance of the ferrous-CO complex using an extinction coefficient of 76 mM Ϫ1 cm Ϫ1 . The individual nNOSoxy and flavoprotein domains were overexpressed and purified as described previously (19,20). The concentrated proteins were stored in a buffer containing 50 mM EPPS, pH 7.5, 2 mM ␤-mercaptoethanol, 10% glycerol, and 0.25 M NaCl.…”

“…Protein Sample Preparation-Ferrous nNOS was prepared by reducing CaM-bound ferric enzyme with NADPH in a gastight cuvette under anaerobic conditions as described previously (19,20). Briefly, a buffered solution containing ϳ120 M nNOS, 10 mM Arg, 2 mM 5MeH 4 B (or 1 mM H 2 B), 200 M CaM, and 600 M Ca 2ϩ was made anaerobic by several cycles of vacuum and flushing with deoxygenated N 2 , and then had N 2 -purged NADPH solution added to give 0.1 to 0.2 mM NADPH.…”

Nitric-oxide Synthase Forms N-NO-pterin and S-NO-Cys

“…Such an allosteric regulatory mechanism is supported by dimer dissociation in iNOS (53) and eNOS (20) upon treatment with NO donors. In contrast, nNOS truncation mutants that lack the ZnS 4 site remain dimeric (54), suggesting this regulatory mechanism is isoform-specific. For its physiological roles in immune function, iNOS generates large bursts of toxic nitrogen oxides (55).…”