Potentiometric Analysis of the Flavin Cofactors of Neuronal Nitric Oxide Synthase

Abstract

Midpoint reduction potentials for the flavin cofactors in the reductase domain of rat neuronal nitric oxide synthase (nNOS) in calmodulin (CaM)-free and -bound forms have been determined by direct anaerobic titration. In the CaM-free form, the FMN potentials are -49 +/- 5 mV (oxidized/semiquinone) -274 +/- 5 mV (semiquinone/reduced). The corresponding FAD potentials are -232 +/- 7, and -280 +/- 6 mV. The data indicate that each flavin can exist as a blue (neutral) semiquinone. The accumulation of blue semiquinone on the FMN is considerably higher than seen on the FAD due to the much larger separation (225 mV) of its two potentials (cf. 48 mV for FAD). For the CaM-bound form of the protein, the midpoint potentials are essentially identical: there is a small alteration in the FMN oxidized/semiquinone potential (-30 +/- 4 mV); the other three potentials are unaffected. The heme midpoint potentials for nNOS [-239 mV, L-Arg-free; -220 mV, L-Arg-bound; Presta, A., Weber-Main, A. M., Stankovich, M. T., and Stuehr, D. J. (1998) J. Am. Chem. Soc. 120, 9460-9465] are poised such that electron transfer from flavin domain is thermodynamically feasible. Clearly, CaM binding is necessary in eliciting conformational changes that enhance flavin to flavin and flavin to heme electron transfers rather than causing a change in the driving force.

Chimeric Enzymes of Cytochrome P450 Oxidoreductase and Neuronal Nitric-oxide Synthase Reductase Domain Reveal Structural and Functional Differences

“…After the flavins have been reduced and catalysis ends, the nNOS reductase domain assumes a partially reduced, air stable semiquinone form (26,32), a state also observed with CYPOR. Eventually, over a period of hours for nNOS and days for CYPOR, the flavins will become fully oxidized.…”

NO formation by a catalytically self-sufficient bacterial nitric oxide synthase from Sorangium cellulosum

“…Formally the 2Fe2S cluster replaces the FMN cofactor in the mammalian NOSs. The redox potentials of FMN in nNOS are Ϫ49 mV (ox/sq, oxidized/semiquinone) and Ϫ274 mV (sq/hq, semiquinone/hydroquinone) (31). In nNOS, FAD has redox potentials of Ϫ232 mV (ox/sq) and Ϫ280 mV (sq/hq) that allow for FMN reduction by FAD.…”

Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase

“…The inability to populate the blue di-semiquinoid form of NOS reductase suggests that there is a kinetic restriction on forming this intermediate. Potentiometric studies of nNOS reductase [22] have indicated that the redox potential of the oxidized FMN (FMN ox )\FMN sq couple, like that of CPR [21], is substantially more positive than the other flavin couples in the domain, and thus should provide a strong driving force for electron transfer to the FMN [i.e. hydroquinone FAD (FAD hq )\FMN ox FAD sq \ FMN sq ].…”

“…Unlike with NOS, however, the cytochrome P450 enzymes are distinct entities that are not covalently linked to the reductase module. CPR and the NOS reductase domain have common functional properties : both transfer electrons to several different artificial acceptors such as cytochrome c, dichlorophenolindophenol, and ferricyanide [14,16,20] and the redox potentials of the flavin couples are similar for each enzyme [21,22]. Also, like CPR, the NOS reductase domain must be reduced beyond a one-electronreduced state to enable efficient electron transfer to the oxygenase domain [23,24].…”

“…Studies with NOS have focused on thermodynamic and rapid kinetic approaches with the aim of establishing the mechanism of electron flow, the role of bound CaM, and any mechanistic equivalence with CPR. CaM binding does not perturb the redox potentials of the flavin couples in nNOS reductase [22], but is proposed to enhance the rates of electron transfer through the reductase domain [16][17][18] and to the oxygenase domain [15]. Moreover, flavin to haem electron transfer is suggested to be rate-limiting on the basis of stoppedflow studies of the three NOS isoforms [24].…”