Two-Dimensional Magnetization Exchange Spectroscopy of Anabaena 7 120 from Amino Acid Residues Surrounding the 2 Fe2 S * Clustert , $ Ferredoxin . Nuclear Overhauser Effect and Electron Self-Exchange Cross Peaks

Hyperfine 'H N M R 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 29,2263-22711 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, Arg4*, 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, 4126-41311. Three chemical exchange cross peaks were detected in magnetization exchange spectra of half-reduced ferredoxin and assigned to the 'Ha 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 peaks in a variety of paramagnetic proteins. I n macromolecules, magnetization exchange by chemical exchange and cross relaxation (the nuclear Overhauser effect or NOE)' are formally equivalent (Noggle & Schirmer, 1971; Neuhaus & Williamson, 1989). The first magnetization exchange investigation of a paramagnetic protein was the classic pulsed NMR study of chemical self-exchange in a mixture of oxidized and reduced cytochrome c (Redfield & Gupta, 1971). Cross-relaxation (NOE) studies of paramagnetically shifted (hyperfine) resonances appeared later (Gordon & Wuthrich, 1978) and became a valuable tool for spectral assignments, particularly in heme proteins (Trewhella et al., 1979; Keller & Wuthrich, 1980; Lecomte et al., 1985). Hyperfine proton signals of iron-sulfur proteins were first reported by Poe et al. (1970). The hyperfine signals of fer'This work was supported by USDA Grant 88-37262-3406 (J.L.M.) and NIH Grant GM39082 (H.M.H.). NMR studies were carried out in the National Magnetic Resonance Facility at Madison, which is supported by NIH Grant RR02301. Equipment in the NMR Facility was purchased with funds from the NIH Biomedical Research Technology Program (Grant RR02301), the University of Wisconsin, the NSF Biological Instrumentation Program (Grant DMB-8415048), NIH Shared Instrumentation Program (Grant RR02781), and the U S . Department of Agriculture. L.S. was supported by a grant from the Norwegian Research Council for Science and the Humanities (NAVF). *NMR data will be deposited in BioMagResBank (Ulrich et al., 1989) under ref ID 889. National Magnetic Resonance Facility at Madison and Biochemistry Department. 11 Present address: University of Trondheim, Kjemisk Institute, Rosenborg, 7055 Dragvoll, Norway. * Present address: Department of Chemistry, University of California, Berkeley, CA 94720. lnstitute for Enzyme Research and Chemistry Department. redoxins generally are characterized by their relative intensities (number of protons) and by the magnitudes and temperature dependencies of their chemical shifts [reviewed by Markley et al. (1986)l. Two new strategies have been used recently to assign ferredoxin hyperfine proton resonances: incorporation of deuterium-labeled cysteine into the protein (Cheng et al., 1990) and detection of one-dimensional (1 D) nuclear Overhauser enhancements (NOES) (Dugad et al., 1990). The latter approach led to preliminary sequence-specific assignments of several of the cysteine proton resonances in the ferredoxins from Spirulina platensis and Prophyra umbilicalis (Dugad et al., 1990). Weak NOE's present in regions of overlapping peaks are difficult to detect in 1 D difference spectra; they should be more easily resolved in 2D NOE spectra. We show here that hyperfine 2D magnetization exchange spectroscopy can be carried out with a ferredoxin provided that short mixing times and rapid recycle times are used in the data collection. Analysis of the patterns of NOE cross peaks from reduced Anabaena 7 120 ferredoxin enabled us to characterize the proton spin systems of all four cysteine ligands to the 2Fe-2S* cluster plus those of three other residues located near the cluster. In addition, chemical exchange cross peaks found in 2D magnetization exchange spectra of half-reduced ferredoxin I Abbreviations: Fd, ferredoxin; FID, free induction decay; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; pH*, pH meter reading (glass electroae calibrated with normal buffers) of a sample dissolved in ZHzO uncorrected for deuterium isotope effects; TI, spin-lattice relaxation time; TPPI, time-proportional phase incrementation, TSP, 3-(trimethylsilyl)propionate. 0006-296019 110430-7363$02.50/0