Evidence for conformational dynamics and molecular aggregation in cytochrome P450 102 (BM-3).
The native molecular weight of affinity-purified cytochrome P450 102 from barbiturate-induced Bacillus megaterium has been studied by sedimentation methods and HPLC size-exclusion chromatography. Sedimentation velocity experiments yielded an s020,w = 9.244 S for the holocytochrome, but the diffusion coefficient was unexpectedly large and varied widely with centrifugal field, ionic strength, and protein concentration. Addition of 50 mM DL-dithiothreitol (DTT) caused a small decrease in the value of s020,w, but D20 still did not behave as expected. The sedimentation coefficients were consistent with a molecular weight of about 200,000, and the diffusion coefficients indicated molecular aggregation. Sedimentation equilibrium analyses suggested that the native enzyme was a mixture of monomer, dimer, trimer, and tetramer. However, after incubation of P450 102 with DTT, sedimentation equilibrium demonstrated that the enzyme was dimeric (molecular weight 236,000). HPLC size-exclusion chromatography of the cytochrome showed the presence of four peaks, which corresponded to 1.45-mer, 2.06-mer, 3.02-mer, and a higher molecular weight fraction; aggregated forms accounted for about 52% of the P450 102. Incubation of the enzyme with DTT caused a shift toward the 1.45-mer, but dimer, trimer, and the high molecular weight peak still persisted; the shift was not attributable to disulfide bond reduction. The 1.45-mer was determined to be a monomeric species of significantly asymmetric geometry. Together, the results indicated that cytochrome P450 exists with monomer, dimer, trimer, etc. in equilibrium, contrary to the expectation that this soluble P450 would be monomeric.(ABSTRACT TRUNCATED AT 250 WORDS)
Black
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Department of Biochemistry, University of Texas Health Center at Tyler 75710-2003.
Martin
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D001410:Bacillus megaterium / Q000201:enzymology; D001426:Bacterial Proteins; D005591:Chemical Fractionation; D016009:Chi-Square Distribution; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase; D010316:Particle Size; D011487:Protein Conformation; D014461:Ultracentrifugation; D014783:Viscosity
D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases