We have developed and employed multiple amino acid-specific isotopic labeling schemes to obtain definitive assignments for active site 1H NMR resonances of iron(II)- and iron(III)-superoxide dismutase (Fe(II)SOD and Fe(III)SOD) from Escherichia coli. Despite the severe relaxivity of high-spin Fe(III), we have been able to assign resonances to ligand His' δ1 protons near 100 ppm, and β and α protons collectively between 20 and 50 ppm, in Fe(III)SOD. In the reduced state, we have assigned all but 7 ligand protons, in most cases residue-specifically. A pair of previously unreported broad resonances at 25.9 and 22.1 ppm has been conclusively assigned to the β protons of Asp 156, superseding earlier assignments (Ming et al. (1994) Inorg. Chem., 33, 83-87). We have exploited higher temperatures to resolve previously unobserved ortho-like ligand His proton resonances, and specific isotopic labeling to distinguish between the possibilities of δ2 and ε1 protons. These are the closest protein protons to Fe(II) and therefore they have the broadest (~4000 Hz) and most difficult to detect resonances. Our assignments permit interpretation of temperature dependences of chemical shifts, pH dependences and H/D exchange rates in terms of a hydrogen bond network and the Fe(II) electronic state. Interestingly, Fe(II)SOD's axial His ligand chemical shifts are similar to those of the axial His ligand of Rhodopseudomonas palustris cytochrome c' (Bertini et al. (1988) Inorg. Chem., 37, 4814-4821) suggesting that Fe(II)SOD's equatorial His2Asp-ligation is able to reproduce some of the electronic, and thus possibly chemical, properties of heme coordination for Fe2+.
|Number of pages||12|
|Journal||Journal of Biomolecular NMR|
|State||Published - 2000|
Bibliographical noteFunding Information:
by the A.C.S., for partial support of this research under ACS-PRF 33266-AC4,3. A.-F.M. is pleased to thank N.I.H. for financial support (GM55210).
- Active site
- Non-heme iron
- Superoxide dismutase
ASJC Scopus subject areas