Hildebrandt P, Greinert R, Stier A, Stockburger M, Taniguchi H
Max-Planck-Institut für Biophysikalische Chemie, Abteilung Spektroskopie, Göttingen, FRG.
FEBS Lett. 1988 Jan 18;227(1):76-80. doi: 10.1016/0014-5793(88)81417-0.
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.
表面增强共振拉曼（SERR）光谱已被用于研究吸附在胶体银悬浮液或银电极上的纯化兔肝细胞色素P-450 LM2血红素的振动光谱。通过与“溶质”物种的共振拉曼（RR）光谱进行比较，证明了SERR技术的高灵敏度。为了确定吸附的P-450的结构和功能完整性，选择了两个不同的特征。在吸附的P-450的SERR光谱以及溶解酶的RR光谱中，都可以观察到氧化型P-450上底物诱导的自旋态变化以及硫醇盐配体对还原型P-450中氧化态标记带v4的影响。这些发现表明，底物结合位点附近的蛋白质结构和硫醇盐的配位作用不受与金属表面相互作用的影响。这两个结构元素对P-450的功能至关重要。因此，通过这种高灵敏度的RR光谱版本可以研究P-450的酶促作用的基本过程。
