Two electron reduced oxygenated intermediate observed in D251N cytochrome P450cam

D. F. Benson, K. S. Snslick, Stephen Sligar

Research output: Contribution to journalArticlepeer-review


The overall rate determining step in cytochrome P450cam catalysis is the reduction of the ferrous dioxygen complex (oxy P50) whcih has made the detection of catalytic intermediates after this rreduction difficult. However, for D251N cytochrome P450cam the overall rate determining step occurs after the reduction of oxy-P450, due to proton transfer possibly being the rate determining step. Putidaredoxin oxidation kinetics demonstrate the transfer of one electron from reduced putidaredoxin to oxy P450 within the first second of the reaction, to form a two elctron reduced oxygenated P450 intermediate. Direct correlation of the decay of this new intermediate with 5-hydroxycamptor production confirms that th is species exists before while being completely coupled to product formation. Various spectroscopic characterizations indicate that this intermediate is prior to O O bond heterolysis, however the P450 heme electronic structure is significantly different from oxy P450. We suggest that this intermediate is a ferrous superoxide heme complex.

Original languageEnglish (US)
JournalFASEB Journal
Issue number9
StatePublished - 1997

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics


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