MULTINUCLEAR, MULTIDIMENSIONAL NMR-STUDIES OF ANABAENA-7120 HETEROCYST FERREDOXIN - SEQUENCE-SPECIFIC RESONANCE ASSIGNMENTS AND SECONDARY STRUCTURE OF THE OXIDIZED FORM IN SOLUTION

Sequence-specific assignments were determined for the diamagnetic proton resonances from recombinant Anabaena 7120 heterocyst ferredoxin (M(r) = 11 000) produced in Escherichia coli. Several samples selectively labeled with nitrogen-15 were prepared for use in two-dimensional heteronuclear multiple quantum coherence (HMQC) [Muller, L. (1979) J. Am. Chem. Soc. 101, 4481-4484] experiments. A sample uniformly labeled with nitrogen-15 was also prepared and used in two three-dimensional experiments: NOESY-HMQC and TOCSY-HMQC [Zuiderweg, E. R. P., & Fesik, S. W. (1989) Biochemistry, 28, 2387-2391; Marion, D., Ikura, M., Tschudin, R., & Bax, A. (1989) J. Magn. Reson. 85, 393-399]. The sequential assignment strategy relied on the detection of N-15-edited interresidue H-1(i)alpha/ H-1(i+1)N NOE connectivities. Starting points and checks were provided by HMQC spectra of the selectively labeled samples. A sample doubly labeled with carbon-13 and nitrogen-15 was also prepared and used in three triple-resonance experiments: HNCA, HNCO, and HN(CO)CA [Ikura, M., Kay, L. E., & Bax, A. (1990) Biochemistry 29, 4659-4667; Kay, L. E., Ikura, M., Tschudin, R., & Bax, A. (1990) J. Magn. Reson. 89, 496-514]. The HNCA and HN(CO)CA spectra, which were used to confirm assignments from NOE connectivities, provided independent sequential assignments from spin couplings. Resonances from 18 residues were not seen in the diamagnetic region of the NMR spectrum. Several of these residues are very close to the [2Fe-2S] cluster, and their absence is explained by paramagnetic broadening and/or shifting. Other of these residues are located in a very flexible region, and the absence of their signals is attributed to exchange effects. Secondary structural features (four beta-sheets and two alpha-helices) were determined purely on the basis of interresidue NOE connectivities. Structural information from NMR is compared to that from the X-ray structure of the same protein [Jacobson, B. L., Chae, Y. K., Markley, J. L., Rayment, I., & Holden, H. M. (1993) Biochemistry 32, 6788-6793] and from the related Anabaena 7120 vegetative ferredoxin. Observed differences in local mobility and structure may be responsible for the differential specificity of the heterocyst and vegetative ferredoxins.