Thermal inactivation of the reductase domain of cytochrome P450 BM3.
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.
Jamakhandi
Arvind P
AP
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, 72205, USA.
Jeffus
Brandon C
BC
Dass
Vandana R
VR
Miller
Grover P
GP
D015086:2,6-Dichloroindophenol / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001671:Biochemistry / Q000379:methods; D002021:Buffers; D002384:Catalysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D019281:Dimerization; D004789:Enzyme Activation; D005486:Flavin Mononucleotide / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D009994:Osmolar Concentration; D010710:Phosphates; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013696:Temperature
D001426:Bacterial Proteins; D002021:Buffers; D003432:Cross-Linking Reagents; D010710:Phosphates; D005486:Flavin Mononucleotide; D045304:Cytochromes c; D003577:Cytochrome P-450 Enzyme System; D015086:2,6-Dichloroindophenol; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases