SURFACE-ENHANCED RESONANCE RAMAN-SCATTERING FROM CYTOCHROMES-P-450 ADSORBED ON CITRATE-REDUCED SILVER SOLS

Four mammalian liver microsomal cytochrome P-450 enzymes were adsorbed onto citrate-reduced silver colloidal particles, and the active site of each was studied by surface-enhanced resonance Raman scattering (SERRS) spectroscopy. There were differences in the spin states of the heme groups in each protein. Methylcholanthrene-induced rat liver cytochrome P-450 IA2 was approximately 90% high spin. Phenobarbital-induced rat liver cytochromes P-450 IIB1 (90% low spin) and P-450 IIB2 (mixed high and low spin) and rabbit liver cytochrome P-450 IIB4 (100% low spin) were shown to be biologically active while adsorbed on the metal surface by following benzphetamine-induced low- to high-spin-state conversion of the heme prosthetic group. On formation of the mixed low- and high-spin-state P-450 IIB4 benzphetamine complex, the low-spin-state marker band upsilon-10 shifts down by 3 cm-1. It is suggested that reorganization of the water molecules in the pocket upon binding of the benzphetamine is responsible. P-450 proteins are readily denatured at a sol surface, and the protein/sol preparation described in this paper is carefully controlled so that the surface of the sol is "biocompatible". The ability of this particular sol to support an active enzyme is due to a layer of citrate ions that form a coating on the sol surface of the enzyme, providing a spacer between the silver and the protein and protecting it from silver-induced reactions. In addition, the heme group is not on the surface of the protein. This fact and the effect of the citrate layer are sufficient to preserve the integrity of the active site, and yet the distance between the silver and the heme is close enough for SERRS. If the sol is carefully prepared, no denaturation involving changing spin state is observed, although denaturation to P-420 can be forced with acid or alkali. Surface selection rules suggest that the heme orientation is less flat relative to the surface of the metal particle than is heme in cytochrome c. Thus, a "biocompatible sol" has been prepared and reactions of the immobilized enzymes at the metal surface have been examined in situ in contact with aqueous media by using only a few nanograms of sample.