Low-Temperature Optical Spectroscopy of Native and Azide-Reacted Bovine Cu,Zn Superoxide Dismutase. A Structural Dynamics Study

Abstract

The optical absorption spectra of native and N(3-)-reacted Cu,Zn superoxide dismutase (SOD) has been studied in the temperature range 300-10 K. The broad d-d bands observed in the room temperature spectrum, centered at 14,700 cm-1 (native enzyme) and at 15,550 cm-1 (N(3-)-reacted enzyme), are clearly split at low temperature into two bands each, centered at 12,835 and 14,844 cm-1 and at 14,418 and 16,300 cm-1, respectively. The thermal behavior of the 23,720 cm-1 band present in the spectrum of the native enzyme indicates that this band belongs to the His61-->Cu(II) ligand to metal charge transfer transition. Analysis of the zeroth, first, and second moments of the various bands as a function of temperature allowed us to obtain useful information on the stereodynamic properties of the metal site in SOD. In particular for the native protein, it was possible to infer a variation in the metal ligand relative position that occurs as the temperature is lowered and that likely involves all of the ligands except His61. On the other hand, the site is stabilized upon N3- binding, and in this case a variation in the metal ligand position is observed only at the level of the bound anion. The possible relation of these properties to the catalytic mechanism of the enzyme is discussed.

Conformational substates of ferricytochrome c revealed by combined optical absorption and electronic circular dichroism spectroscopy at cryogenic temperature

“…From the spectroscopic point of view, the statistical substates contribute to the rather large spectral Gaussian widths of protein absorption bands (3-5), while taxonomic substates are responsible for their sub-bands structure, like e.g. in the case of the near infrared charge transfer bands of deoxyhemoglobin, deoxymyoglobin and superoxide dismutase (6,7), and of the infrared stretching band of CO bound to myoglobin (2,8,9). Protein conformational substates have substantial functional relevance, since they are responsible for the heterogeneity observed in many dynamical processes like the binding of ligands to the active site; a role of conformational substates in protein folding/unfolding processes has also been suggested (10-…”

Spectroscopic Characterization of Recombinant Cu,Zn Superoxide Dismutase from Photobacterium leiognathi Expressed in Escherichia coli: Evidence for a Novel Catalytic Copper Binding Site

“…The spectrum of the copper chromophore of the bacterial enzyme displays two bands with maxima at 680 and at 407 nm, respectively. The broad band centered at 680 nm has been demonstrated in the bovine enzyme to be actually composed by the partial overlap of two components which are due to the d-d transitions of the copper atom (Cupane et al, 1994). This band in the P. leiognathi enzyme has an extinction coefficient slightly higher ( ) 320 M -1 cm -1 ) than that found in the bovine enzyme ( ) 300 M -1 cm -1 ).…”

Cupricyclins, Novel Redox-Active Metallopeptides Based on Conotoxins Scaffold

“…The difference spectrum is also shown to evidence the appearance of a band at 300–312 nm, indicative of a Cu 2+ -histidine charge-transfer [21], [22].…”

“…Optical spectroscopy studies demonstrated that, also in the case of Cupricyclin-2, addition of a stoichiometric amount of CuCl 2 induces the appearance of absorption bands with maxima at 300–312 nm and 520–600 nm which, by analogy with Cupricyclin-1, were attributed to a Cu 2+ -histidine charge-transfer band and to electronic transitions of copper d-d orbitals typical of copper complexes with nitrogen ligands, respectively [22], [23]. The calculated molar extinction coefficient at 525 nm was 476 M −1 cm −1 .…”

“…Addition of a stoichiometric amount of CuCl 2 to Cupricyclin-1 (0.6 mM final concentration) induced the appearance of absorption bands with maxima at 300–312 nm and 520–600 nm (Figure 5), the first indicative of a Cu 2+ -histidine charge-transfer [22], the second due to electronic transitions of copper d-d orbitals and typical of copper complexes with nitrogen ligands [22], [23]. The calculated molar extinction coefficient of holo-Cupricyclin-1 at 595 nm is 203 M −1 cm −1 .…”