ASSIGNMENT OF ACTIVE-SITE PROTONS IN THE H-1-NMR SPECTRUM OF REDUCED HUMAN CU/ZN SUPEROXIDE-DISMUTASE

600-MHz H-1-NMR and nuclear Overhauser enhancement spectroscopy (NOESY) spectra in (H2O)-H-2 and (H2O)-H-2, as well as truncated driven NOE difference spectra in H2O of reduced human Cu(II)2Zn(II)2 superoxide dismutase (Cu/Zn SOD) have been recorded and used to assign the active-site proton signals. A derivative with histidines selectively deuteriated in the C2 position has been used for the detection of the HC2 histidine protons. 16 out of 17 observed signals of the 18 active-site histidine ring protons have been assigned. The results are compared with previous proposals based on more limited data sets. The numerous cross peaks confirm that the structure in solution is essentially similar to the crystallographic data obtained on the oxidized form. Probably this holds also for His63 which in the reduced form is not bridging any more the two metal ions. The effects of azide binding on the exchangeable H-1-NMR signals of the reduced protein are also reported.