Role of Asp¹³⁹³in Catalysis, Flavin Reduction, NADP(H) Binding, FAD Thermodynamics, and Regulation of the nNOS Flavoprotein

Abstract

Nitric oxide synthases (NOS) are flavoheme enzymes with important roles in biology. The reductase domain of neuronal NOS (nNOSr) contains a widely conserved acidic residue (Asp(1393)) that is thought to facilitate hydride transfer between NADPH and FAD. Previously we found that the D1393V and D1393N mutations lowered the NO synthesis activity and the rates of heme and flavin reduction in full-length nNOS. To examine the mechanisms for these results in greater detail, we incorporated D1393V and D1393N substitutions into nNOSr along with a truncated NADPH-FAD domain construct (FNR) and characterized the mutants. D1393V nNOSr had markedly lower (<or=1000x) cytochrome c reductase, ferricyanide reductase, and NADPH oxidase activities than the wild type. D1393N nNOSr also had lower reductase activities (<or=10x) but had greater NADPH oxidase activity than that of the wild type, as did its FNR fragment. Both mutants had an altered interaction between FAD and the nicotinamide ring of NADP(+), slower flavin reduction by NADPH, altered FAD midpoint potentials, a normal CaM response, and, in one case (D1393N), faster flavin oxidation by O(2) and a lack of FMN shielding in response to NADPH binding. The results suggest that the two mutants have compromised catalysis for two different reasons. In D1393V nNOSr, hydride transfer from NADPH to FAD is so slow that it compromises all downstream electron-transfer events. In D1393N nNOSr, the increased oxidation of reduced flavins by O(2) and thermodynamic destabilization of the FAD semiquinone uncouples or limits electron transfer to an extent that it inhibits downstream catalysis. These effects are due in part to the mutations eliminating (D1393V) or altering (D1393N) the native side-chain hydrogen-bonding properties of Asp(1393) as well as removing its negative charge.

Catalytic Reduction of a Tetrahydrobiopterin Radical within Nitric-oxide Synthase

“…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.…”

“…The EPR spectral properties of the flavin semiquinone radical (FSQ) were recorded for the nNOS flavoprotein domain (20) and for the H 2 B-bound, nNOS fulllength protein (24). The FSQ radical was generated in either protein by adding a slight molar excess of NADPH to each protein in air-saturated buffer and then waiting for the flavins to air oxidize, while monitoring FSQ formation in a UV-visible spectrophotometer as a broad peak centered near 600 nm (20). This procedure results in both proteins containing an air-stable FMN semiquinone radical (20,24).…”

Differences in a Conformational Equilibrium Distinguish Catalysis by the Endothelial and Neuronal Nitric-oxide Synthase Flavoproteins

“…The kinetic traces were fit to a single-exponential decay function to yield the observed NADP ϩ or S-NADP ϩ dissociation rate (k off ). Redox Potentiometry-Sample preparation and redox titrations were carried out in a glove box (Bell Technology) under nitrogen atmosphere with oxygen levels below 5 ppm as described previously (41). The NOSr protein concentration was 30 -40 M containing either EDTA (1 mM) or CaCl 2 (2 mM) ϩ CaM (60 -80 M) in buffer A. Absorption spectra were recorded in Cary 50 using a dip probe detector, and the potentials were monitored using Accumet AB15 coupled to a silver/ silver chloride electrode saturated with 4 M KCl.…”

“…Both proteins were excited at 457 nm wavelength, and the fluorescence spectra were recorded from 480 to 650 nm. The isolated FNR subdomain was prepared as described previously (41). The fluorescence intensities of the FNR subdomain in buffer A were also measured under similar conditions.…”