UV RESONANCE RAMAN SCATTERING FROM METAL-COORDINATING HISTIDINE RESIDUES IN CU, ZN-SUPEROXIDE DISMUTASE
1998
Metal-binding modes of histidine residues in Cu,Zn-superoxide dismutase (Cu,Zn-SOD) were investigated by UV resonance Raman spectroscopy. The metal-bridging imidazolate (Im-) ring of His61 gives prominent Raman bands at ca. 1565, 1335, 1285, 1260, 1050 and 980 cm-1. On the other hand, the imidazole ring of other histidines gives a Raman band at ca. 1395 or 1355 cm-1 when it is ligated to a metal ion via the Nτ or Nπ atom, respectively, and the other nitrogen atom is deuterated (ImD). These Raman marker bands together with the previously established correlation between the C4=C5 stretching wavenumber and metal-coordination site for non-deuterated imidazole (ImH) were used to investigate the metal-binding modes of histidine residues in the reduced and inhibitor-bound states of Cu,Zn-SOD. Upon reduction of the enzyme, the Raman bands of the metal-bridging Im- ring disappear. Concomitantly, a new band appears at 1590 cm-1, which is assignable to the C4=C5 stretch of an ImH ring coordinating to a metal ion via the Nπ atom. These Raman spectral changes provide direct evidence that the Cu—Nτ linkage of His61 is broken upon reduction and the Nτ atom is protonated in the reduced state. The binding of a competitive inhibitor (CN-) produces wavenumber downshifts of Raman bands arising from vibrations of the Nτ—C5=C4 moiety of the His61 Im- ring, indicating a structural modification of His61 by the inhibitor binding. © 1998 John Wiley & Sons, Ltd.
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