Thermal inactivation of the reductase domain of cytochrome P450 BM3

• Published in: Archives of biochemistry and biophysics 2005-07, Vol.439 (2), p.165-174
• Main Authors: Jamakhandi, Arvind P., Jeffus, Brandon C., Dass, Vandana R., Miller, Grover P.
• Format: Article
• Language: English
• Subjects: 2,6-Dichloroindophenol - metabolism; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Biochemistry - methods; BM3; BMR; Buffers; Catalysis; Cross-Linking Reagents - chemistry; Cytochrome; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - metabolism; Cytochromes c - metabolism; Dimerization; Domain; Enzyme Activation; Flavin Mononucleotide - metabolism; Inactivation; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - metabolism; NADPH-Ferrihemoprotein Reductase; Osmolar Concentration; P450; Phosphates; Protein Denaturation; Protein Structure, Tertiary; Reductase; Temperature; Thermal stability
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2005.04.022

Although the reductase domain of cytochrome P450 BM3 (BMR) catalyzes the reduction of cytochrome c and 2,6-dichlorophenolindophenol, we observed a catalytically independent loss of activity. By varying the incubation time for the enzyme prior to reaction initiation, we measured an inactivation rate of 0.22 min −1. We hypothesized that either an active BMR dimer dissociates to an inactive monomer or BMR undergoes denaturation. We were not able to trap or destabilize a dimer, and BMR inactivation proved to be irreversible. Addition of excess FMN only slightly decreased the rate of inactivation from 0.22 to 0.13 min −1, indicating inactivation likely does not reflect loss of flavin. When inactivation rates as a function of temperature were fit to the Arrhenius equation, the energy required to inactivate BMR was 9.9 kcal mol −1—equivalent to a few hydrogen bonds. The potential instability of BMR under certain conditions raises concerns for the use of BMR as a model or surrogate P450 reductase in other systems.