Beta sheet 2-alpha helix C loop of cytochrome P450 reductase serves as a docking site for redox partners.
As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein-protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1*CPR complex and inactive (CYP2B1)(2)*CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge-charge interactions between CPR and complex partners.
School of Biological Sciences and Technology and Hormone Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea.
Hyun-Hee
HH
Jang
Arvind P
AP
Jamakhandi
Shane Z
SZ
Sullivan
Chul-Ho
CH
Yun
Paul F
PF
Hollenberg
Grover P
GP
Miller
D019943:Amino Acid Substitution; D000818:Animals; D002384:Catalysis; D002417:Cattle; D019362:Cytochrome P-450 CYP2B1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045304:Cytochromes c / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006736:Horses; D008958:Models, Molecular; D020125:Mutation, Missense; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000502:physiology; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D055672:Static Electricity
D045304:Cytochromes c; D019362:Cytochrome P-450 CYP2B1; D009251:NADPH-Ferrihemoprotein Reductase