Evidence for retention of biological activity of a non-heme iron enzyme adsorbed on a silver colloid: a surface-enhanced resonance Raman scattering study.
The structure and catalytic properties of the enzyme (E) chlorocatechol dioxygenase (CCD) adsorbed on a citrate-reduced silver colloid are analyzed by surface-enhanced resonance Raman spectroscopy (SERRS). This is the first SERRS study of a non-heme metalloenzyme. It is demonstrated that the native conformation of CCD is retained in the adsorbed state by comparison of resonance Raman scattering (RRS) from CCD in solution with SERRS from CCD adsorbed on the silver colloid. Both spectra show clear evidence of vibrational bands typical of iron-tyrosinate proteins. Furthermore, it is demonstrated that adsorbed CCD retains 60-85% of its enzymatic activity in the reaction of catechol substrate (S) with O2 to give the dioxygenated product (P) cis,cis-muconate. This is accomplished by enzyme assays of Ag-adsorbed CCD and comparison of the SERRS of Ag-adsorbed enzyme-substrate (ES) complex under anaerobic conditions with that of Ag-adsorbed ES in the presence of dioxygen. The SERRS difference spectrum, ES(aerobic)--ES(anaerobic), shows clear evidence for the appearance of the vibrational modes of adsorbed product. The analogous SERR difference spectroscopy experiment is also carried out for the enzyme-inhibitor (EI) complex of CCD with tetrachlorocatechol (TCC). Slow turnover of CCD-TCC is observed by SERRS on exposure to dioxygen which is consistent with the slow rate of turnover of TCC by CCD in solution.
Broderick
J B
JB
Department of Chemistry, Northwestern University, Evanston, Illinois 60208-3113.
Natan
M J
MJ
O'Halloran
T V
TV
Van Duyne
R P
RP
D000327:Adsorption; D002384:Catalysis; D002396:Catechols / Q000737:chemistry; D003102:Colloids; D049308:Dioxygenases; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism*; D019159:Nonheme Iron Proteins; D010105:Oxygenases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D012834:Silver; D012996:Solutions; D013059:Spectrum Analysis, Raman
D002396:Catechols; D003102:Colloids; D008667:Metalloproteins; D019159:Nonheme Iron Proteins; D012996:Solutions; D012834:Silver; D010105:Oxygenases; D049308:Dioxygenases; C053101:catechol oxygenase II