Geometries and electronic structures of cyanide adducts of the non-heme iron active site of superoxide reductases: vibrational and ENDOR studies.
We have added cyanide to oxidized 1Fe and 2Fe superoxide reductase (SOR) as a surrogate for the putative ferric-(hydro)peroxo intermediate in the reaction of the enzymes with superoxide and have used vibrational and ENDOR spectroscopies to study the properties of the active site paramagnetic iron center. Addition of cyanide changes the active site iron center in oxidized SOR from rhombic high-spin ferric (S = 5/2) to axial-like low-spin ferric (S = 1/2). Low-temperature resonance Raman and ENDOR data show that the bound cyanide adopts three distinct conformations in Fe(III)-CN SOR. On the basis of 13CN, C15N, and 13C15N isotope shifts of the Fe-CN stretching/Fe-C-N bending modes, resonance Raman studies of 1Fe-SOR indicate one near-linear conformation (Fe-C-N angle approximately 175 degrees) and two distinct bent conformations (Fe-C-N angles <140 degrees). FTIR studies of 1Fe-SOR at ambient temperatures reveals three bound C-N stretching frequencies in the oxidized (ferric) state and one in the reduced (ferrous) state, indicating that the conformational heterogeneity in cyanide binding is a characteristic of the ferric state and is not caused by freezing-in of conformational substates at low temperature. 13C-ENDOR spectra for the 13CN-bound ferric active sites in both 1Fe- and 2Fe-SORs also show three well-resolved Fe-C-N conformations. Analysis of the 13C hyperfine tensors for the three substates of the 2Fe-SOR within a simple heuristic model for the Fe-C bonding gives values for the Fe-C-N angles in the three substates of ca. 123 degrees (C3) and 133 degrees (C2), taking a reference value from vibrational studies of 175 degrees (C1 species). Resonance Raman and ENDOR studies of SOR variants, in which the conserved glutamate and lysine residues in a flexible loop above the substrate binding pocket have been individually replaced by alanine, indicate that the side chains of these two residues are not involved in direct interaction with bound cyanide. The implications of these results for understanding the mechanism of SOR are discussed.
Clay
Michael D
MD
Department of Chemistry, University of Georgia, Athens, Georgia 30602, USA.
Yang
Tran-Chin
TC
Jenney
Francis E
FE
Kung
Irene Y
IY
Cosper
Christopher A
CA
Krishnan
Rangan
R
Kurtz
Donald M
DM
Adams
Michael W W
MW
Hoffman
Brian M
BM
Johnson
Michael K
MK
D001665:Binding Sites; D002942:Circular Dichroism; D003486:Cyanides / Q000737:chemistry* / Q000378:metabolism*; D016969:Desulfovibrio vulgaris / Q000201:enzymology; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D008958:Models, Molecular; D019159:Nonheme Iron Proteins / Q000737:chemistry / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D020027:Pyrococcus furiosus / Q000201:enzymology; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D013481:Superoxides / Q000737:chemistry / Q000378:metabolism
D003486:Cyanides; D019159:Nonheme Iron Proteins; D013481:Superoxides; D010088:Oxidoreductases; C121849:superoxide reductase