Open AccessDetermination of the redox potentials and electron transfer properties of the FAD‐ and FMN‐binding domains of the human oxidoreductase NR1
Author(s) -
Finn Robert D.,
Basran Jaswir,
Roitel Olivier,
Wolf C. Roland,
Munro Andrew W.,
Paine Mark J. I.,
Scrutton Nigel S.
Publication year - 2003
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1033.2003.03474.x
Subject(s) - semiquinone , chemistry , redox , flavin group , electron transfer , oxidoreductase , photochemistry , reductase , flavin adenine dinucleotide , stereochemistry , biochemistry , cofactor , enzyme , inorganic chemistry
Human novel reductase 1 (NR1) is an NADPH dependent diflavin oxidoreductase related to cytochrome P450 reductase (CPR). The FAD/NADPH‐ and FMN‐binding domains of NR1 have been expressed and purified and their redox properties studied by stopped‐flow and steady‐state kinetic methods, and by potentiometry. The midpoint reduction potentials of the oxidized/semiquinone (−315 ± 5 mV) and semiquinone/dihydroquinone (−365 ± 15 mV) couples of the FAD/NADPH domain are similar to those for the FAD/NADPH domain of human CPR, but the rate of hydride transfer from NADPH to the FAD/NADPH domain of NR1 is ≈ 200‐fold slower. Hydride transfer is rate‐limiting in steady‐state reactions of the FAD/NADPH domain with artificial redox acceptors. Stopped‐flow studies indicate that hydride transfer from the FAD/NADPH domain of NR1 to NADP + is faster than hydride transfer in the physiological direction (NADPH to FAD), consistent with the measured reduction potentials of the FAD couples [midpoint potential for FAD redox couples is −340 mV, cf −320 mV for NAD(P)H]. The midpoint reduction potentials for the flavin couples in the FMN domain are −146 ± 5 mV (oxidized/semiquinone) and −305 ± 5 mV (semiquinone/dihydroquinone). The FMN oxidized/semiquinone couple indicates stabilization of the FMN semiquinone, consistent with (a) a need to transfer electrons from the FAD/NADPH domain to the FMN domain, and (b) the thermodynamic properties of the FMN domain in CPR and nitric oxide synthase. Despite overall structural resemblance of NR1 and CPR, our studies reveal thermodynamic similarities but major kinetic differences in the electron transfer reactions catalysed by the flavin‐binding domains.