An examination of the cyanide derivative of bovine superoxide dismutase with electron-nuclear double resonance

The Cu(II) sites of native, azido- and cyano-derivatives of bovine superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) have been examined by electron-nuclear double resonance ENDOR). The ENDOR spectrum of the native protein taken at the gII extreme shows resolved structure due to the file directly coordinated N-atoms of the histidine ligands. These spectra are too complex for interpretation but suggest inequivalent coupling between the electronic spin and the four ligand N-atoms. By contrast, the azido protein reveals one type of nitrogen with well-resolved hyperfine and quadrupole splittings (Azz = 37.9+/- 1 MHz, Pzz = 1.54+/-0.02 MHz), and the cyano form reveals one well-resolved set of nitrogen lines (Azz =47.8 +/-0.4 MHz, Pzz =1.62+/-0.01 MHz) and one type of partially resolved nitrogen (Azz =37.0+/-1 MHz). The cyano form also reveals a complex spectrum in the low-frequency domain (1-10 MHz). Through isotopic substitution and computer simulation, the spectrum is shown to be a composite of the ENDOR from the remote imidazole nitrogens and the cyanide nitrogen. The component of the hyperfine constant perpendicular to the C14N bond axis is AN[perpendicular] =3.9+/-0.3 MHz and along the bond axis is AN[perpendicular] [approximate, equal]5.7 MHz. The quadrupole interaction appears to be greatest along the CN axis with Qz'z' = 1.0+/-0.1 MHz and Qx'x'y'y' [approximate]0. Based on an analysis of the hyperfine and quadrupole interactions seen at two extremes of the electron paramagnetic spectrum, we propose a square-planar arrangement of three imidazole nitrogen and one CN- carbon around the copper. Within this plane two imidazole nitrogens are strongly coupled and magnetically equivalent, the third is inequivalent (slightly weaker hyperfine interaction) and forms a trans relationship with the cyanide. This model is consistent with other observations on the cyano-derivative.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23979/1/0000228.pd