Abstract
Nitric-oxide synthases (NOSs) are flavo-heme enzymes whose electron transfer reactions are controlled by calmodulin (CaM). The NOS flavoprotein domain includes a ferredoxin-NADP(+) reductase (FNR)-like module that contains NADPH- and FAD-binding sites. FNR-like modules in related flavoproteins have three conserved residues that regulate electron transfer between bound NAD(P)H and FAD. To investigate the function of one of these residues in neuronal NOS (nNOS), we generated and characterized mutants that had Val, Glu, or Asn substituted for the conserved Asp-1393. All three mutants exhibited normal composition, spectral properties, and binding of cofactors, substrates, and CaM. All had slower NADPH-dependent cytochrome c and ferricyanide reductase activities, which were associated with proportionally slower rates of NADPH-dependent flavin reduction in the CaM-free and CaM-bound states. Rates of NO synthesis were also proportionally slower in the mutants and were associated with slower rates of CaM-dependent ferric heme reduction. However, a D1393V mutant whose flavins had been prereduced with NADPH had a normal rate of heme reduction. This indicated that the kinetic defect was restricted to flavin reduction step(s) in the mutants and suggested that this limited their catalytic activities. Together, our results show the following. 1) The presence and positioning of the Asp-1393 carboxylate side chain are critical to enable NADPH-dependent reduction of the nNOS flavoprotein. 2) Control of flavin reduction is important because it ensures that the rate of heme reduction is sufficiently fast to enable NO synthesis by nNOS.
Highlights
Nitric-oxide synthases (NOSs) are flavo-heme enzymes whose electron transfer reactions are controlled by calmodulin (CaM)
Flavin analysis showed that the mutants contained normal quantities of FAD and FMN (ϳ1 each per neuronal NOS (nNOS) heme)
Their ability to bind and elute from the 2Ј,5Ј-ADP resin during purification showed that they still could reversibly bind NADPH. These properties indicate that substitution of Asp-1393 by Val, Glu, or Asn did not grossly alter the physical properties of nNOS
Summary
Materials—All reagents and materials were obtained from Sigma or sources previously reported [16, 42, 43]. Steady-state NADPH Oxidation—Wild-type and mutant nNOS enzymes were diluted to 4 M in a cuvette that contained buffer, H4B, Ca2ϩ, CaM, and other additives as used for the NO synthesis assay [42], except that here oxyhemoglobin, L-Arg, and NADPH were omitted. The prereduced enzyme was transferred into the stopped-flow device and mixed under anaerobic conditions with a CO-saturated solution containing 1 mM Ca2ϩ and 4 M CaM to trigger heme reduction. Proteins were irradiated with 460 nm light using an in-line 8% filter, and the emission at 530 nm was monitored versus time before and after consecutive addition of 1 mM Ca2ϩ and 3 mM EDTA
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
