Characterization of hydride transfer to flavin adenine dinucleotide in neuronal nitric oxide synthase reductase domain: Geometric relationship between the nicotinamide and isoalloxazine rings
Rt. Miller et Ap. Hinck, Characterization of hydride transfer to flavin adenine dinucleotide in neuronal nitric oxide synthase reductase domain: Geometric relationship between the nicotinamide and isoalloxazine rings, ARCH BIOCH, 395(1), 2001, pp. 129-135
Based on the similarity in both structure and function of the reductase dom
ain of neuronal nitric oxide synthase (nNOSred) to that of NADPH-cytochrome
P450 reductase (CPR), we determined whether the characteristics of hydride
transfer from NADPH to flavin adenine dinucleotide (FAD) were similar for
both proteins. Secondly, we questioned whether hydride transfer from NADPH
to either nNOSred or holo-nNOS was rate limiting for reactions catalyzed by
these two proteins. Utilizing 500 MHz proton NMR and deuterated substrate,
we determined that the stereospecificity of hydride transfer from NADPH an
d the conformation of the nicotinamide ring around the glycosidic bond were
similar between CPR and nNOSred. Specifically, nNOSred abstracts the A-sid
e hydrogen from NADPH, and the nicotinamide ring is in the anti conformatio
n. We determined that the rate of hydride transfer to FAD appears to become
partially rate limiting only for exceptionally good electron acceptors suc
h as cytochrome c. Hydride transfer is not rate limiting for NO. production
under any conditions used in this study. Interestingly, the deuterium isot
ope effect was decreased in the cytochrome c reductase assay with both nNOS
and nNOSred when the assays were conducted in high ionic strength buffer,
suggesting an increase in the rate of hydride transfer to FAD. These result
s are in stark contrast to results obtained with CPR (D. S. Sem, and C. B.
Kasper, 1995, Biochemistry 34, 3391-3398) whereby hydride transfer is parti
ally rate limiting at high, but not at low, ionic strength. The seemingly o
pposite results in deuterium isotope effect observed with CPR and nNOSred,
under conditions of high and low ionic strength, suggest differences in str
ucture and/or regulation of these important flavoproteins. (C) 2001 Academi
c Press.