Surface enhanced resonance Raman study of phenobarbital-induced rabbit liver cytochrome P-450 LM2.
Surface enhanced resonance Raman (SERR) spectroscopy has been used to study the vibrational spectra of the heme of purified rabbit liver cytochrome P-450 LM2 which was adsorbed on colloidal silver suspensions or on a silver electrode. Bases on a comparison with the resonance Raman (RR) spectra of the 'solute' species the high sensitivity of the SERR technique is demonstrated. Two different features were chosen in order to determine the structural and functional integrity of the adsorbed P-450. Both, substrate-induced spin state changes on the oxidized P-450 and the effect of the thiolate ligand on the oxidation state marker band v4 in the reduced P-450 could be observed in the SERR spectra of the adsorbed as well as in the RR spectra of the dissolved enzyme. These findings indicate that the protein structure near the substrate binding site and the coordination by thiolate are not affected by the interaction with the metal surface. Both structural elements are crucial for the function of P-450. Thus the elementary processes of the enzymatic action of P-450 can be investigated by this highly sensitive version of RR spectroscopy.
Hildebrandt
P
P
Max-Planck-Institut für Biophysikalische Chemie, Abteilung Spektroskopie, Göttingen, FRG.
Greinert
R
R
Stier
A
A
Stockburger
M
M
Taniguchi
H
H
D000327:Adsorption; D000818:Animals; D003102:Colloids; D003577:Cytochrome P-450 Enzyme System; D003580:Cytochromes; D004566:Electrodes; D006418:Heme; D008081:Liposomes / Q000378:metabolism; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D017137:Nonoxynol; D010084:Oxidation-Reduction; D010634:Phenobarbital / Q000494:pharmacology; D011092:Polyethylene Glycols; D011817:Rabbits; D012834:Silver; D012995:Solubility; D013059:Spectrum Analysis, Raman
D003102:Colloids; D003580:Cytochromes; D008081:Liposomes; D017137:Nonoxynol; D012834:Silver; D011092:Polyethylene Glycols; D006418:Heme; C013464:cytochrome P420; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital