The role of tryptophan 97 of cytochrome P450 BM3 from Bacillus megaterium in catalytic function. Evidence against the 'covalent switching' hypothesis of P-450 electron transfer.
The 'Covalent Switching' hypothesis suggests that a strongly conserved tryptophan residue acts as a mediator of electron-transfer flow between redox partners in cytochrome P-450 systems [Baldwin, Morris and Richards (1991) Proc. R. Soc. London B 245, 43-51]. We have investigated the effect of alteration of the conserved tryptophan (Trp-97) in cytochrome P-450 BM3 (P-450 102) from Bacillus megaterium. Replacement of Trp-97 with Ala, Phe or Tyr results in a decrease in the natural haem content and alters the resting spin state of the remaining haem in the purified mutant enzymes. However, kinetic analyses indicate that the mutant enzymes retain high levels of catalytic activity. C.d. and e.p.r. spectroscopy also reveal little alteration in secondary structure or change in the pattern of haem ligation. These findings cast doubt on the covalent switching mechanism of intermolecular electron flow in the P-450s, but indicate that this residue plays a role in the association of the haem prosthetic group.
Department of Biochemistry, University of Glasgow, U.K.
A W
AW
Munro
K
K
Malarkey
J
J
McKnight
A J
AJ
Thomson
S M
SM
Kelly
N C
NC
Price
J G
JG
Lindsay
J R
JR
Coggins
J S
JS
Miles
D000409:Alanine / Q000235:genetics / Q000502:physiology; D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins; D001483:Base Sequence; D002384:Catalysis; D002942:Circular Dichroism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D009251:NADPH-Ferrihemoprotein Reductase; D010649:Phenylalanine / Q000235:genetics / Q000502:physiology; D017433:Protein Structure, Secondary; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000235:genetics / Q000502:physiology*; D014443:Tyrosine / Q000235:genetics / Q000502:physiology
D001426:Bacterial Proteins; D017931:DNA Primers; D014443:Tyrosine; D006418:Heme; D010649:Phenylalanine; D014364:Tryptophan; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D000409:Alanine