Long distance charge redistribution upon Cu,Zn-superoxide dismutase reduction: significance for dismutase function.
Cu,Zn-superoxide dismutase (Cu,Zn-SOD) is a ubiquitous enzyme with an essential role in antioxidant defense. To better understand structural factors at the origin of the highly efficient superoxide dismutation mechanism, we analyzed the consequence of copper reduction on the electronic properties of the backbone and individual amino acids by using electrochemistry coupled to Fourier transform infrared spectroscopy. Comparison of data recorded with bovine erythrocyte and recombinant chloroplastic Cu,Zn-SOD from Lycopersicon esculentum, expressed as a functional tetramer in Escherichia coli and (14)N- or fully (15)N-labeled, demonstrated that the infrared changes were dominated by reorganizations of peptide bonds and histidine copper ligands. Two main infrared modes of histidine side chain, markers of metal coordination, were identified by using Cu- and Zn-methylimidazole models: the nu(C(4)C(5))at 1605-1594 cm(-1) or approximately 1586 cm(-1) for Ntau or Npi coordination, and the nu(C(5)Ntau) at approximately 1113-1088 cm(-1). These modes, also identified in Cu,Zn-SOD by using (15)N labeling, showed that the electronic properties of the histidine Ntau ligands of copper are mostly affected upon copper reduction. A striking conclusion of this work is that peptide groups from loops and beta-sheet largely participate in charge redistribution upon copper reduction, and in contrast, electronic properties of polar and charged amino acids of the superoxide access channel remain unaffected. This is notably shown for the strictly conserved Arg-143 by site-directed mutagenesis on chloroplastic Cu,Zn-SOD. Charge compensation by the peptide backbone and preserved electronic properties of the superoxide access channel and docking site upon copper reduction may be the determinant factors for the high reaction kinetics of superoxide with both reduced and oxidized Cu,Zn-SOD.
Commissariat à l'Energie Atomique/Cadarache, Laboratoire de Bioénergétique Cellulaire, UMR 6191 CNRS-CEA-Aix-Marseille II, Université de la Méditerranée CEA-1000, Bāt. 156, F-13108 Saint-Paul-lez-Durance, France.
François
F
Dupeyrat
Claude
C
Vidaud
Alain
A
Lorphelin
Catherine
C
Berthomieu
D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D003300:Copper / Q000737:chemistry; D004563:Electrochemistry; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry; D018551:Solanum lycopersicum / Q000201:enzymology; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000737:chemistry / Q000378:metabolism; D014867:Water / Q000737:chemistry
D007093:Imidazoles; D010940:Plant Proteins; D014867:Water; D013912:Threonine; D006639:Histidine; D003300:Copper; D001120:Arginine; D013482:Superoxide Dismutase