Skip to main content
U.S. flag

An official website of the United States government

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.

Structural Alterations of the Heme Environment of Cytochrome P450cam and the Y96F Mutant as Deduced by Resonance Raman Spectroscopy



G Niaura, Vytas Reipa, M P. Mayhew, Marcia J. Holden, V L. Vilker


Resonance Raman spectroscopy at 2.5 cm-1 resolution was used to probe differences in wild type and Y96F mutant P450cam (CYP101), both with and without bound camphor or styrene substrates. In the substrate-free state, the spin state equilibrium is shifted from 6-coordinate low spin (6CLS) toward more 5-coordinate high spin (5CHS) when tyrosine 96 in the substrate pocket is replaced by phenylalanine. About 25% of substrate-free Y96F mutant is 5CHS as opposed to 8% for substrate-free WT P450cam. Spin equilibrium constants calculated from Raman intensities indicate that the thermodynamic driving force for the high spin formation is closely related to the hydrogen bond formation between substrate and the tyrosine 96 residue, and that the driving force for electron transfer from putidaredoxin, the natural redox partner of P450cam, is significantly smaller upon styrene binding than for camphor binding. Upon binding of camphor, the substrate interaction of the 6th ligand in the active site of low spin Y96F results in shrinkage of the heme core by 0.004 D due to the iron movement out of porphyrin plane. Spectral differences also suggest that there is a tilt in camphor toward the pyrrole III ring upon Y96F mutation. This finding is consistent with the altered product distribution found for camphor hydroxylation by the Y96F mutant relative to the single enantiomer produced by the WT enzyme. Styrene perturbed the RR spectra relative to the substrate-free state to a lesser degree than camphor for both WT and Y96F. This suggests that styrene imposes less stress on the macrocycle ring than does camphor.
Archives of Biochemistry and Biophysics
No. 1


cytochrome P450cam, Resonance Raman Spectroscopy, Y96F


Niaura, G. , Reipa, V. , Mayhew, M. , Holden, M. and Vilker, V. (2003), Structural Alterations of the Heme Environment of Cytochrome P450cam and the Y96F Mutant as Deduced by Resonance Raman Spectroscopy, Archives of Biochemistry and Biophysics (Accessed July 23, 2024)


If you have any questions about this publication or are having problems accessing it, please contact

Created December 31, 2002, Updated October 12, 2021