Investigation of the low frequency dynamics of heme proteins: Native and mutant cytochrome P450cam and redox partner complexes

Venugopal Karunakaran, Ilia Denisov, Stephen G. Sligar, Paul M. Champion

Research output: Contribution to journalArticlepeer-review

Abstract

Vibrational coherence spectroscopy (VCS) is used to investigate the low-frequency dynamics of camphor-free and camphor-bound cytochrome P450 cam (CYP 101) and its L358P mutant. The low-frequency heme vibrations are found to be perturbed upon binding to the electron transfer partner putidaredoxin (Pdx). A strong correlation between the "detuned" vibrational coherence spectrum, which monitors frequencies between 100 and 400 cm-1, and the lower frequency part of the Raman spectrum is also demonstrated. The very low frequency region ≤200 cm-1, uniquely accessed by open-band VCS measurements, reveals a mode near 103 cm-1 in P450cam when camphor is not present in the distal pocket. This reflects the presence of a specific heme distortion, such as saddling or ruffling, in the substrate-free state where water is coordinated to the low-spin iron atom. Such distortions are likely to retard the rate of electron transfer to the substrate-rree protein. The presence or strong mode near ∼33 cm -1in the camphor-bound form suggests a significant heme-doming distortion, which is supported by analysis using normal coordinate structural decomposition. Pdx also displays a strong coherent vibration near 30 cm 1 that in principle could be involved in vibrational resonance with its electron transfer target. A splitting of the 33 cm-1 feature and intensification of a mode near 78 cm-1 appear when the P450 cam/Pdx complex is formed. These observations are consistent with vibrational mixing and heme geometric distortions upon Pdx binding that are coincident with the increased thiolate electron donation to the heme. The appearance of a mode near 65 cm-1 in the coherence spectra of the L358P mutant is comparable to the mode at 78 cm-1 seen in the P450cam/Pdx complex and is consistent with the view that the heme and its environment in the L358P mutant are similar to the Pdx-bound native protein. Resonance Raman spectra are presented for both P450cam and the L358P mutant and the changes are correlated with an increased amount of thiolate electron donation to the heme in the mutant sample.

Original languageEnglish (US)
Pages (from-to)5665-5677
Number of pages13
JournalJournal of Physical Chemistry B
Volume115
Issue number18
DOIs
StatePublished - May 12 2011

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Surfaces, Coatings and Films
  • Materials Chemistry

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