Interaction of nitrogen monoxide with cytochrome P-450 monitored by surface-enhanced resonance Raman scattering

Area of Science:

• Biochemistry
• Spectroscopy
• Enzymology

Background:

• Mammalian cytochrome P-450 2B4 is crucial in metabolizing various compounds.
• Nitrogen monoxide (NO) is known to inhibit cytochrome P-450 activity.
• Understanding the mechanism of NO interaction with P-450 is vital for pharmacology and toxicology.

Purpose of the Study:

• To elucidate the in situ reaction mechanism of cytochrome P-450 2B4 with nitrogen monoxide and oxygen.
• To characterize the structural and electronic changes in the P-450 active site during NO exposure.
• To investigate the formation of nitrated byproducts and their relation to enzyme inhibition.

Main Methods:

• Surface-enhanced resonance Raman scattering (SERRS) spectroscopy was employed for high-sensitivity, in situ analysis.
• Mammalian cytochrome P-450 2B4 was reacted with controlled concentrations of nitrogen monoxide and oxygen.
• Spectroscopic data were analyzed to determine haem coordination, spin state, and amino acid modifications.

Main Results:

• An initial six-coordinate, low-spin haem-nitrogen monoxide adduct was observed.
• A subsequent irreversible reaction formed a five-coordinate, high-spin iron (III) haem, with NO dissociation.
• Nitration of a nearby aromatic side chain, likely tyrosine, was detected, linked to biphasic enzyme inhibition.

Conclusions:

• SERRS provides definitive in situ information on cytochrome P-450 active site dynamics.
• The reaction proceeds via NO adduct formation, haem modification, and subsequent nitration of tyrosine residues.
• This mechanism explains the observed biphasic inhibition of P-450 enzymes by NO.