Carbon monoxide binding to cytochrome P450(BM-3): Evidence for a substrate-dependent conformational change

M. A. McLean, H. Yeom, S. G. Sligar

Research output: Contribution to journalArticlepeer-review

Abstract

The kinetics of carbon monoxide binding to cytochrome P450(BM-3) in the presence and absence of substrate has been investigated using flash photolysis. The second order kinetics for CO association with the substrate-free form of the protein appear biphasic. Deconvolution into two exponentials yields fast and slow rate constants of 11.1 ± 0.6 x 106 M-1 s-1 and 3.5 ± 0.2 x 106 M-1 s-1, respectively with 52% of the signal being attributed to the fast phase. Interestingly, upon binding of a substrate such as laurate, the second order kinetics become mono phasic, with a value of 3.5 x 1066 M-1 s-1, which are similar to the slow rate found in the substrate-free form of the protein. We have also examined the geminate CO rebinding kinetics in the presence and absence of various substrates. In the substrate-free form the overall geminate yield is 30%, and addition of a substrate increases the geminate yield to roughly 50%. Both the substrate-free and substrate-bound forms exhibit complex geminate kinetics which cannot be described by a simple three-state kinetic model. Extension of this model to include four states is required. The addition of substrate causes an increase in the geminate rate constants resulting in a larger geminate amplitude when compared to the substrate-free form. There is also evidence for a correlation between the volume occupied by the substrate and the geminate rate constants. These results are discussed in terms of substrate-dependent conformational changes in cytochrome P450(BM-3) and the overall energy landscape of the hemoprotein which couples to conformer equilibria.

Original languageEnglish (US)
Pages (from-to)700-705
Number of pages6
JournalBiochimie
Volume78
Issue number8-9
DOIs
StatePublished - 1996

Keywords

  • Carbon monoxide binding
  • Hemoprotein
  • P450(BM-3)

ASJC Scopus subject areas

  • Biochemistry

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