15N-NMR characterization of His residues in and around the active site of FeSOD.
We have exploited (15)N-NMR to observe histidine (His) side chains in and around the active site of Fe-containing superoxide dismutase (FeSOD). In the oxidized state, we observe all the non-ligand His side chains and in the reduced state we can account for all the signals in the imidazole spectral region in terms of the non-ligand His', paramagnetically displaced signals from two backbone amides, and the side chain of glutamine 69 (Gln69). We also observe signals from the His' that ligate Fe(II). These confirm that neither the Q69H nor the Q69E mutation strongly affects the Fe(II) electronic structure, despite the 250 mV and >660 mV increases in E(m) they produce, respectively. In the Q69H mutant, we observe two new signals attributable to the His introduced into the active site in place of Gln69. One corresponds to a protonated N and the other is strongly paramagnetically shifted, to 500 ppm. The strong paramagnetic effects support the existence of an H-bond between His69 and the solvent molecule coordinated to Fe(II), as proposed based on crystallography. Based on previous information that His69 is neutral, we infer that the shifted N is not protonated. Therefore, we propose that this N represents a site of H-bond acceptance from coordinated solvent, representing a reversal of the polarity of this H-bond from that in WT (wild-type) FeSOD protein. We also present evidence that substrate analogs bind to Fe(II)SOD outside the Fe(II) coordination sphere, affecting Gln69 but without direct involvement of His30.
Miller
Anne-Frances
AF
Dept. Chemistry, University of Kentucky, Lexington, KY 40506-0055, USA. afm@uky.edu
Yikilmaz
Emine
E
Vathyam
Surekha
S
D001665:Binding Sites; D020134:Catalytic Domain; D005290:Ferric Compounds / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013482:Superoxide Dismutase / Q000737:chemistry*
D005290:Ferric Compounds; D006639:Histidine; D013482:Superoxide Dismutase