Molecular dissection of human methionine synthase reductase: determination of the flavin redox potentials in full-length enzyme and isolated flavin-binding domains.
Human methionine synthase reductase (MSR) catalyzes the NADPH-dependent reductive methylation of methionine synthase. MSR is 78 kDa flavoprotein belonging to a family of diflavin reductases, with cytochrome P450 reductase (CPR) as the prototype. MSR and its individual flavin-binding domains were cloned as GST-tagged fusion proteins for expression and purification from Escherichia coli. The isolated flavin domains of MSR retain UV-visible and secondary structural properties indicative of correctly folded flavoproteins. Anaerobic redox titrations on the individual domains assisted in assignment of the midpoint potentials for the high- and low-potential flavin. For the isolated FMN domain, the midpoint potentials for the oxidized/semiquinone (ox/sq) couple and semiquinone/hydroquinone (sq/hq) couple are -112 and -221 mV, respectively, at pH 7.0 and 25 degrees C. The corresponding couples in the isolated FAD domain are -222 mV (ox/sq) and -288 mV (sq/hq). Both flavins form blue neutral semiquinone species characterized by broad absorption peaks in the long-wavelength region during anaerobic titration with sodium dithionite. In full-length MSR, the values of the FMN couples are -109 mV (ox/sq) and -227 mV (sq/hq), and the corresponding couple values for FAD are -254 mV (ox/sq) and -291 mV (sq/hq). Separation of the MSR flavins does not perturb their thermodynamic properties, as midpoint potentials for all four couples are similar in isolated domains and in full-length MSR. The redox properties of MSR are discussed in relation to other members of the diflavin oxidoreductase family and the mechanism of electron transfer.
Wolthers
Kirsten R
KR
Department of Biochemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom.
Basran
Jaswir
J
Munro
Andrew W
AW
Scrutton
Nigel S
NS
D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers / Q000737:chemistry; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000235:genetics / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008745:Methylation; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D011199:Potentiometry; D011993:Recombinant Fusion Proteins; D014466:Ultraviolet Rays
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; C111241:methionine synthase reductase; D005287:Ferredoxin-NADP Reductase