Journal article
Substrate Dependent Native Luminescence from Cytochromes P450 3A4, 2C9, and P450cam
The journal of physical chemistry. B, Vol.120(12), pp.3038-3047
03/31/2016
Handle:
https://hdl.handle.net/2376/103683
PMCID: PMC5048684
PMID: 26939024
Abstract
Metalloporphyrin containing proteins, such as cytochrome P450, play a key role in biological systems. The spectroscopic properties of metalloporphyrins have been a subject of intense interest and intense debate for over 50 years. Iron-porphyrins are usually believed to be nonfluorescent. Herein we report that, contrary to this belief, cytochrome P450 heme groups luminesce with enough intensity to be of use in the characterization of these enzymes. To confirm that the emission is from the heme, we destroyed the heme by titration with cumene hydroperoxide and measured the changes in emission upon titration with compounds known to bind to the distal face of the heme in two human cytochrome P450 enzymes, known as CYP3A4 and CYP2C9. The titration curves gave spectral dissociation constants that were not significantly different from those reported using the Soret UV/vis absorbance changes. We have tentatively assigned the broad luminescence at ∼500 nm to a (1)ππ* → gs fluorescence and the structured luminescence above 600 nm to a (3)ππ* → gs phosphorescence. These assignments are not associated with the Q-band, and are in violation of Kasha's rule. To illustrate the utility of the emission, we measured spectral dissociation constants for testosterone binding to P450 3A4 in bilayers formed on glass coverslips, a measurement that would be very difficult to make using absorption spectroscopy. Complementary experiments were carried out with water-soluble P450cam.
Metrics
8 Record Views
Details
- Title
- Substrate Dependent Native Luminescence from Cytochromes P450 3A4, 2C9, and P450cam
- Creators
- Carlo Barnaba - Washington State University, Department of Chemistry, PO Box 644630, Pullman, WA, USA, 99164-4630Sara C Humphreys - Washington State University, Department of Chemistry, PO Box 644630, Pullman, WA, USA, 99164-4630Adam O Barden - Washington State University, Department of Chemistry, PO Box 644630, Pullman, WA, USA, 99164-4630Jeffrey P Jones - Washington State University, Department of Chemistry, PO Box 644630, Pullman, WA, USA, 99164-4630James A Brozik - Washington State University, Department of Chemistry, PO Box 644630, Pullman, WA, USA, 99164-4630
- Publication Details
- The journal of physical chemistry. B, Vol.120(12), pp.3038-3047
- Academic Unit
- Chemistry, Department of
- Publisher
- United States
- Grant note
- R01 GM114369 / NIGMS NIH HHS R01 GM110790 / NIGMS NIH HHS GM114396 / NIGMS NIH HHS GM110790 / NIGMS NIH HHS
- Identifiers
- 99900546620201842
- Language
- English
- Resource Type
- Journal article