Multinuclear magnetic resonance studies of the 2Fe.cntdot.2S* ferredoxin from Anabaena species strain PCC 7120. 3. Detection and characterization of hyperfine-shifted nitrogen-15 and hydrogen-1 resonances of the oxidized form

Abstract

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.(ABSTRACT TRUNCATED AT 250 WORDS)

The tautomeric state of histidines in myoglobin

“…A comparison of the nitrogen chemical shifts in the metcyano and metaquo forms, and with other protein shifts, indicates that the nitrogen chemical shift is negligibly affected by paramagnetic effects. This has been observed in the iron-sulfur clusters as well (Oh and Markley, 1990) and is expected for the shift experienced by a nucleus with a low magnetogyric ratio when the number of bonds to the paramagnetic center is large enough to prevent contact contribution. The only strongly deviating nitrogen shift is for the proximal histidine, where contact shift is present.…”

NMR Spectroscopy of Paramagnetic Metalloproteins

“…[133] However, a few groups continued to deepen the understanding of the effects of hyperfine coupling in NMR spectroscopy and simultaneously to exploit technological advancements to increase the signal-to-noise ratio of paramagnetic-system spectra. [134][135][136][137][138][139][140][141][142][143] Eventually, a protocol for solving the structure of a paramagnetic metalloprotein was reported in 1994 [144] and since then many examples have appeared in the literature. [63,145] Of course, every metal ion has its own peculiarities and should be regarded as a case by itself.…”

NMR structures of paramagnetic metalloproteins

“…The sensitivity of 15 N direct-detected experiments presently achieved is, however, quite low and constitutes a limitation. Nevertehless, cases have been described in which 15 N-detected experiments have been successfully used in paramagnetic proteins (Oh & Markley, 1990 ;Xia et al 1995 ;Wilkens et al 1998b ;Caillet et al 2006).…”