Electron transfer by human wild-type and A287P mutant P450 oxidoreductase assessed by transient kinetics: functional basis of P450 oxidoreductase deficiency.
Cytochrome P450 oxidoreductase (POR) is a 2-flavin protein that transfers electrons from NADPH via its FAD and FMN moieties to all microsomal cytochrome P450 enzymes, including steroidogenic and drug-metabolizing P450s. Defects in the POR gene can cause POR deficiency (PORD), manifested clinically by disordered steroidogenesis, genital anomalies and skeletal malformations. We examined the POR mutant A287P, which is the most frequent cause of PORD in patients of European ancestry and partially disrupts most P450 activities in vitro. Flavin content analysis showed that A287P is deficient in FAD and FMN binding, although the mutation site is distant from the binding sites of both flavins. Externally added flavin partially restored the cytochrome c reductase activity of A287P, suggesting that flavin therapy may be useful for this frequent form of PORD. Transient kinetic dissection of the reaction of POR with NADPH and the reduction in cytochrome c by POR using stopped-flow techniques revealed defects in individual electron transfer steps mediated by A287P. A287P had impaired ability to accept electrons from NADPH, but was capable of a fast FMN → cytochrome c electron donation reaction. Thus the reduced rates of P450 activities with A287P may be due to deficient flavin and impaired electron transfer from NADPH.
*Center of Excellence in Environmental Toxicology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, PA 19104-6160, U.S.A.
Yi
Y
Jin
*Center of Excellence in Environmental Toxicology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, PA 19104-6160, U.S.A.
Mo
M
Chen
*Center of Excellence in Environmental Toxicology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, PA 19104-6160, U.S.A.
Trevor M
TM
Penning
‡Department of Pediatrics, University of California, San Francisco, San Francisco, California, CA 94143, U.S.A.
Walter L
WL
Miller
D019943:Amino Acid Substitution; D045304:Cytochromes c / Q000378:metabolism; D004579:Electron Transport; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013053:Spectrophotometry
D050505:Mutant Proteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; D045304:Cytochromes c; D009251:NADPH-Ferrihemoprotein Reductase