We aim to extend our understanding of the structure and function of cytochrome P450 and other heme protein systems by using pump-probe kinetics and time resolved resonance Raman spectroscopy over a wide dynamic range in time (ps-ms). Development of a unique temporally stabilized, and electronically tunable, two-color picosecond laser system will enable both the transient Raman and the kinetic studies. Femtosecond coherence spectroscopy (FCS), which is a direct and unique probe of the coherent protein/cofactor vibrational motion induced by electronic transitions associated with biochemical reactions, will also be utilized in studies of the biochemical reaction coordinates as well as the global protein response. One specific hypothesis to be tested addresses the reason behind the anomalously low femtosecond quantum yield for O2 photolysis and the potential for a transient side-on oxygen binding geometry that arises because of the participation of ligand bending modes in the reaction coordinate for dissociation. Vibrationally hot six coordinate oxymyoglobin species have already been detected using white light continuum probes, and we propose to extend such studies to the P450 system and its catalytic intermediates as well as to expand the probe capability to include picosecond Raman scattering techniques. Experiments at low temperature will also be carried out using newly developed optical techniques. These experiments will include the first picosecond and femtosecond studies of geminate ligand recombination as a function of temperature. Exploratory work with the Raman microscope will continue with spatially specific probes of mitochondria and with studies of P450 embedded in membrane nanodiscs. Overall this project has a wide range of health related implications involving cytochrome P450 in particular and heme proteins in general. Many chemotherapeutic agents as well as polycyclic carcinogens are metabolized by P450 systems. Moreover, all metabolic disorders involving a P450 protein must relate to this research, since a fundamental understanding of the active site structure and function of P450 at the molecular level will result in a better insight for those concerned with treatment of these disorders at any level.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK035090-24
Application #
7162122
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Sechi, Salvatore
Project Start
1984-06-01
Project End
2008-11-30
Budget Start
2006-12-01
Budget End
2007-11-30
Support Year
24
Fiscal Year
2007
Total Cost
$314,849
Indirect Cost
Name
Northeastern University
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
001423631
City
Boston
State
MA
Country
United States
Zip Code
02115
Karunakaran, Venugopal; Sun, Yuhan; Benabbas, Abdelkrim et al. (2014) Investigations of the low frequency modes of ferric cytochrome c using vibrational coherence spectroscopy. J Phys Chem B 118:6062-70
Sun, Yuhan; Zeng, Weiqiao; Benabbas, Abdelkrim et al. (2013) Investigations of heme ligation and ligand switching in cytochromes p450 and p420. Biochemistry 52:5941-51
Zeng, Weiqiao; Sun, Yuhan; Benabbas, Abdelkrim et al. (2013) Investigations of ferric heme cyanide photodissociation in myoglobin and horseradish peroxidase. J Phys Chem B 117:4042-9
Sun, Yuhan; Karunakaran, Venugopal; Champion, Paul M (2013) Investigations of the low-frequency spectral density of cytochrome c upon equilibrium unfolding. J Phys Chem B 117:9615-25
Benabbas, Abdelkrim; Karunakaran, Venugopal; Youn, Hwan et al. (2012) Effect of DNA binding on geminate CO recombination kinetics in CO-sensing transcription factor CooA. J Biol Chem 287:21729-40
Karunakaran, Venugopal; Denisov, Ilia; Sligar, Stephen G et al. (2011) Investigation of the low frequency dynamics of heme proteins: native and mutant cytochrome P450(cam) and redox partner complexes. J Phys Chem B 115:5665-77
Barabanschikov, Alexander; Demidov, Alexander; Kubo, Minoru et al. (2011) Spectroscopic identification of reactive porphyrin motions. J Chem Phys 135:015101
Karunakaran, Venugopal; Benabbas, Abdelkrim; Youn, Hwan et al. (2011) Vibrational coherence spectroscopy of the heme domain in the CO-sensing transcriptional activator CooA. J Am Chem Soc 133:18816-27
Karunakaran, Venugopal; Benabbas, Abdelkrim; Sun, Yuhan et al. (2010) Investigations of low-frequency vibrational dynamics and ligand binding kinetics of cystathionine beta-synthase. J Phys Chem B 114:3294-306
Benabbas, Abdelkrim; Ye, Xiong; Kubo, Minoru et al. (2010) Ultrafast dynamics of diatomic ligand binding to nitrophorin 4. J Am Chem Soc 132:2811-20

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