title: Proximal FAD histidine residue influences interflavin electron transfer in cytochrome P450 reductase and methionine synthase reductase.
abstract: Cytochrome P450 reductase (CPR) and methionine synthase reductase (MSR) transfer reducing equivalents from NADPH to FAD to FMN. In CPR, hydride transfer and interflavin electron transfer are kinetically coupled steps, but in MSR the two catalytic steps are represented by two distinct kinetic phases leading to transient formation of the FAD hydroquinone. In human CPR, His(322) forms a hydrogen-bond with the highly conserved Asp(677), a member of the catalytic triad. The catalytic triad is present in MSR, but Ala(312) replaces the histidine residue. To examine if this structural variation accounts for differences in their kinetic behavior, reciprocal substitutions were created. Substitution of His(322) for Ala in CPR does not affect the rate of NADPH hydride transfer or the FAD redox potentials, but does impede interflavin electron transfer. For MSR, swapping Ala(312) for a histidine residue resulted in the kinetic coupling of hydride and interflavin electron transfer, and eliminated the formation of the FAD hydroquinone intermediate. For both enzymes, placement of the His residue in the active site weakens coenzyme binding affinity. The data suggest that the proximal FAD histidine residue accelerates proton-coupled electron transfer from FADH2 to the higher potential FMN; a mechanism for this catalytic role is discussed.
authors.affiliation: Department of Chemistry, University of British Columbia Okanagan, 3333 University Way, Kelowna, BC V1V 1V7, Canada.
authors.forename: Carla E
authors.initials: CE
authors.lastname: Meints
authors.affiliation: Department of Chemistry, University of British Columbia Okanagan, 3333 University Way, Kelowna, BC V1V 1V7, Canada.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Parke
authors.affiliation: Department of Chemistry, University of British Columbia Okanagan, 3333 University Way, Kelowna, BC V1V 1V7, Canada. Electronic address: kirsten.wolthers@ubc.ca.
authors.forename: Kirsten R
authors.initials: KR
authors.lastname: Wolthers
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000737:chemistry / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary
chemical_list: D005182:Flavin-Adenine Dinucleotide; D006639:Histidine; D005486:Flavin Mononucleotide; C111241:methionine synthase reductase; D005287:Ferredoxin-NADP Reductase; D009251:NADPH-Ferrihemoprotein Reductase
keywords: Cytochrome P450 reductase; Electron transfer; Flavin; Methionine synthase reductase; Stopped-flow spectroscopy