Resonance Raman investigations of cytochrome P450(cam) complexed with putidaredoxin

Masashi Unno, James F. Christian, David E. Benson, Nancy C. Gerber, Stephen G. Sligar, Paul M. Champion

Research output: Contribution to journalArticlepeer-review


We have performed resonance Raman and optical absorption studies on ferric cytochrome P450(cam) complexed with oxidized putidaredoxin. Optical absorption and resonance Raman measurements demonstrate that this complexation shifts the spin-state equilibrium of P450(cam) to the low-spin form. In the resonance Raman spectra, the v3 heme skeletal mode characteristic of low-spin P450(cam) intensifies upon complexation with putidaredoxin. Its frequency is indistinguishable to that of the usual low-spin species, indicating that this low-spin form originates from a water-bound, hexacoordinate heme. This observation suggests that putidaredoxin binding affects the distal side of P450(cam) and allows water entry into the heme pocket. We also examined the effects of putidaredoxin binding on the heme axial ligand (Fe-S) stretching mode. The binding of putidaredoxin upshifts the mode by ~3 cm-1. Investigations of the Fe-S mode for the Thr252 → Ala distal pocket mutant and P450(cam) bound to various camphor analogues demonstrate that the frequency of the υ(Fe-S) is sensitive to distal pocket water. The water lowers the Fe-S stretching frequency by ~1 cm-1, demonstrating that the putidaredoxin-induced ~3 cm-1 increase in the Fe-S stretching frequency is not simply caused by the increased hydration of the heme pocket. We also found that the Fe-S frequency is increased by ~0.5 cm-1 in the presence of high salt concentrations. This result suggests that the shielding of positive charges at the proximal face of P450(cam) leads to an increased υ(Fe-S) frequency. The salt effect is consistent with the observation that the binding of negatively charged putidaredoxin at the proximal side of the enzyme also increases the υ(Fe-S) frequency. Taken together, these results strongly suggest that electrostatic shielding of charged groups on the proximal face of P450(cam) takes place when putidaredoxin binds and contributes to the observed upshift of the Fe-S mode.

Original languageEnglish (US)
Pages (from-to)6614-6620
Number of pages7
JournalJournal of the American Chemical Society
Issue number28
StatePublished - Jul 16 1997

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry


Dive into the research topics of 'Resonance Raman investigations of cytochrome P450(cam) complexed with putidaredoxin'. Together they form a unique fingerprint.

Cite this