All the nitrogen signals from the amino acid side chains and 80 of the total of 98 backbone nitrogen signals of the oxidized form of the 2Fe•2S* ferredoxin from Anabaena sp. strain PCC 7120 were assigned by means of a series of heteronuclear two-dimensional experiments [Oh, B.-H., Mooberry, E. S., & Markley, J. L. (1990) Biochemistry (second paper of three in this issue)]. Two additional nitrogen signals were observed in the one-dimensional 15N NMR spectrum and classified as backbone amide resonances from residues whose proton resonances experience paramagnetic broadening. The one-dimensional 15N NMR spectrum shows nine resonances that are hyperfine shifted and broadened. From this inventory of diamagnetic nitrogen signals and the available X-ray coordinates of a related ferredoxin [Tsukihara, T., Fukuyama, K., Nakamura, M., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., Hase, T., & Matsubara, H. (1981) J. Biochem. 90, 1763–1773], the resolved hyperfine-shifted 15N peaks were attributed to backbone amide nitrogens of the nine amino acids that share electrons with the 2Fe•2S* center or to backbone amide nitrogens of two other amino acids that are close to the 2Fe•2S* center. The seven 15N signals that are missing and unaccounted for probably are buried under the envelope of amide signals. 1H NMR signals from all the amide protons directly bonded to the seven missing and nine hyperfine-shifted nitrogens were too broad to be resolved in conventional 2D NMR spectra. From their dependence on the magnetogyric ratio, a 1H resonance should be up to 100 times broader than a 15N resonance that experiences a similar hyperfine interaction. This appears to be the reason why more well-resolved hyperfine-shifted 15N resonances were observed than corresponding 1H resonances. The results suggest that hyperfine-shifted 15N peaks can provide a unique window on the electronic structure and environment of this and other paramagnetic centers. © 1990, American Chemical Society. All rights reserved.
