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. © 2005 Elsevier Inc. All rights reserved.
Jamakhandi, A. P., Jeffus, B. C., Dass, V. R., & Miller, G. P. (2005). Thermal inactivation of the reductase domain of cytochrome P450 BM3. Archives of Biochemistry and Biophysics, 439(2), 165–174. https://doi.org/10.1016/j.abb.2005.04.022