N-15-NMR characterization of His residues in and around the active site of FeSOD

作者：Miller Anne Frances^*; Yikilmaz Emine; Vathyam Surekha

来源：Biochimica et Biophysica Acta-Proteins and Proteomics, 2010, 1804(2): 275-284.

DOI：10.1016/j.bbapap.2009.11.009

• N-15-NMR
• Fe-superoxide dismutase
• Paramagnetic
• Chemical shift
• Redox tuning
• Histidine

We have exploited N-15-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 (FeSOD)-S-II outside the Fe-II Coordination sphere, affecting Gln69 but without direct involvement of His30.

• 出版日期2010-2