EVIDENCE FOR RETENTION OF BIOLOGICAL-ACTIVITY OF A NONHEME IRON ENZYME ADSORBED ON A SILVER COLLOID - A SURFACE-ENHANCED RESONANCE RAMAN-SCATTERING STUDY

The structure and catalytic properties of the enzyme (E) chlorocatecho l dioxygenase (CCD) adsorbed on a citrate-reduced silver colloid are a nalyzed by surface-enhanced resonance Raman spectroscopy (SERRS). This is the first SERRS study of a non-heme metalloenzyme. It is demonstra ted that the native conformation of CCD is retained in the adsorbed st ate by comparison of resonance Raman scattering (RRS) from CCD in solu tion with SERRS from CCD adsorbed on the silver colloid. Both spectra show clear evidence of vibrational bands typical of iron-tyrosinate pr oteins. Furthermore, it is demonstrated that adsorbed CCD retains 60-8 5% of its enzymatic activity in the reaction of catechol substrate (S) with O2 to give the dioxygenated product (P) cis,cis-muconate. This i s accomplished by enzyme assays of Ag-adsorbed CCD and comparison of t he SERRS of Ag-adsorbed enzyme-substrate (ES) complex under anaerobic conditions with that of Ag-adsorbed ES in the presence of dioxygen. Th e SERRS difference spectrum, ES(aerobic) - ES(anaerobic), shows clear evidence for the appearance of the vibrational modes of adsorbed produ ct. The analogous SERR difference spectroscopy experiment is also carr ied out for the enzyme-inhibitor (EI) complex of CCD with tetrachloroc atechol (TCC). Slow turnover of CCD-TCC is observed by SERRS on exposu re to dioxygen which is consistent with the slow rate of turnover of T CC by CCD in solution.