2-dimensional magnetization exchange spectroscopy of Anabaena 7120 ferredoxin- Nuclear Overhauser effect and electron self-exchange cross peaks from amino acid residues surrounding the 2Fe-2S-star cluster

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Hyperfine H-1 NMR signals of the 2Fe-2S* vegetative ferredoxin from Anabaena 7120 have been studied by two-dimensional (2D) magnetization exchange spectroscopy. The rapid longitudinal relaxation rates of these signals required the use of very short nuclear Overhauser effect (NOE) mixing times (0.5-20 ms). The resulting pattern of NOE cross-relaxation peaks when combined with previous 1D NOE results [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271] led to elucidation of the carbon-bound proton spin systems from each of the four cysteines ligated to the 2Fe-2S* cluster in the reduced ferredoxin. Additional NOE cross peaks were observed that provide information about other amino acid residues that interact with the iron-sulfur cluster. NOE cross peaks were assigned tentatively to Leu27, Arg42, and Ala43 on the basis of the X-ray coordinates of oxidized Anabaena 7120 ferredoxin [Rypniewski, W. R., Breiter, D. R., Benning, M. M., Wesenberg, G., Oh, B.-H., Markley, J. L., Rayment, I., & Holden, H. M. (1991) Biochemistry 30, 4126-4131]. Three chemical exchange cross peaks were detected in magnetization exchange spectra of half-reduced ferredoxin and assigned to the H-1-alpha protons of Cys49 and Cys79 [both of whose sulfur atoms are ligated to Fe(III)] and Arg42 (whose amide nitrogen is hydrogen-bonded to one of the inorganic sulfurs of the 2Fe-2S* cluster). The chemical exchange cross peaks provide a means of extending assignments in the spectrum of reduced ferredoxin to assignments in the spectrum of the oxidized protein. Our results suggest that 2D magnetization exchange spectroscopy employing short mixing times will be useful for the assignment and characterization of hyperfine H-1 peaks in a variety of paramagnetic proteins.
Publisher
AMER CHEMICAL SOC
Issue Date
1991-07
Language
English
Article Type
Note
Keywords

SPECIES STRAIN PCC-7120; 2FE.2S-STAR FERREDOXIN; 2-IRON FERREDOXINS; MOLECULAR-CLONING; SEQUENCE-ANALYSIS; OXIDIZED FORM; RESONANCE; SPECTRA; H-1; NMR

Citation

BIOCHEMISTRY, v.30, no.30, pp.7363 - 7368

ISSN
0006-2960
DOI
10.1021/bi00244a002
URI
http://hdl.handle.net/10203/67276
Appears in Collection
BS-Journal Papers(저널논문)
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