The Solution Structure Refinement of the Paramagnetic Reduced High‐Potential Iron‐Sulfur Protein I from Ectothiorhodospira Halophila by Using Stable Isotope Labeling and Nuclear Relaxation

Abstract

The reduced high-potential iron sulfur protein I from Ectothiorhodospira halophila which contains the [4Fe-4SI2+ polymetallic center has been fully labeled with "N and 13C. The protein is paramagnetic, the nuclear relaxation times of nuclei close to the paramagnetic ion are drastically shortened and some strategic dipolar connectivities are lost. Notwithstanding, the solution structure has been reported [Banci, L., Bertini, I., Eltis, L. D., Felli, I. C., Kastrau, D. H. W., Luchinat, C., Piccioli, M., Pierattelli, R. & Smith, M. (1994) Eul: J. Biochem. 225, 715-7251. We have performed classical HNHA, HNCA soft-COSY, soft-HCCH E. COSY and ''N-'H correlated NOESY experiments in order to obtain a set of 3J scalar coupling constants. Some experiments have been optimized to counterbalance the effect of paramagnetism. From heteronuclear single-quantum experiments preceded by a 180" pulse and variable delay times, the non-selective magnetization recovery has been followed from which the contribution to dipolar relaxation of nuclei due to the interaction with the paramagnetic metal ions (pp"'") has been estimated. Finally, the intensities of NOEs have been corrected for the presence of paramagnetic metal ions and these corrected values together with 3J values and ppara data have been used to obtain a well defined solution structure. The aim is that of obtaining a structure with enough constraints to be well resolved all over the protein, including the vicinity of the paramagnetic metal cluster, which is anchored to the protein through the ppsril constraints. In total, 1226 corrected NOESY crosspeaks (of which 945 were found to be meaningful), 37 one-dimensional NOEs, 39 3JHNH,z and 37 3JHNc (providing 45 4 dihedral angle constraints) 54 3JHuHB and 31 3JNHp (providing 26 x, dihedral angle constraints), 4 x2 dihedral angle constraints of the coordinated cysteines, obtained from the hyperfine shifts of the PCH protons, and 58 pa'& constraints, have been used for structure calculation. Restrained molecular dynamics simulations have also been performed to provide the final family of structures. This research demonstrates that stable isotope labeling provides specific advantages for the NMR investigation of paramagnetic molecules, as the small magnetic moment of heteronuclei minimizes the paramagnetic influence of unpaired electrons.Keywords: iron-sulfur; NMR; nuclear relaxation; solution structure; heteronucleus. Abbreviations. HiPIP, High-potential iron-sulfur protein; HMQC, heteronuclear multiple-quantum coherence spectroscopy : HSQC, heteronuclear single-quantum coherence spectroscopy ; GARP, ;I composite pulse decoupling scheme; TPPI, time proportional phase increments ; E. COSY, exclusive correlation spectroscopy ; DIANA, distance geometry algorithm for NMR applications ; PRETTIDIANA, paramagnetic relaxation times to improve DIANA; RMSD, root-mean-square deviation; REM, restrained energy minimization ; RMD, restrained molecular dynamics; REDAC, redundant dihedral angle constraints; DG, distance geometry; ID, 2D and 3D, ...

The metal reductase activity of some multiheme cytochromes c : NMR structural characterization of the reduction of chromium(VI) to chromium(III) by cytochrome c 7

“…However, in such systems the binding of chromium was found often to occur at multiple sites. The use of paramagnetic relaxation for solution structure determination of metalloproteins had been explored by our laboratory (23,24) and had been used in the presence of mobile paramagnetic tags (52,53). The present results obtained on Cyt c 7 become quite relevant, because they provide the key for the understanding on a structural basis the chromium(VI)-reductase activity of the whole class of multiheme cytochromes.…”

“…The proportionality constant k can be found experimentally through calibration by treating line broadening as an NOE-derived upper distance limit. To determine upper distance limits between the chromium(III) and protein protons, we used an approach similar to the Caliba procedure designed by Wüth-rich and coworkers to calibrate 1 H-1 H dipolar constraints (22) and extended to the calibration of longitudinal nonselective relaxation times of protons by Bertini et al (23,24). The procedure used in the present case can be summarized as follows:…”

The solution structure of paramagnetic metalloproteins

“…More generally, each paramagnetic metal ion will have a different Kvalue [28]. The experimental nuclear relaxation rate is the sum of the above mentioned paramagnetic contribution (Eqn 2) plus a diamagnetic contribution [21,29,30]. A procedure was suggested to estimate the average diamagnetic contribution, which is subtracted from the experimental rate.…”

“…The protein contains a [Fe 4 S 4 ] 2 cluster, which is slightly paramagnetic (m eff per Fe 0.85 BM) [22], but the protons of the metal-coordinated cysteines relax very fast (the b protons have T 1 s of the order of 5±10 ms) [23]. About 13 meaningful NOEs per residue were obtained together with a total of 45 constraints for the backbone f dihedral angle (from 3 J HNHa and 3 J HNC H values), and 26 constraints for the side chain x 1 dihedral angle (from 3 J HaHb and 3 J NHb values) [21]. Dihedral angle constraints were estimated also for the Fe-Sg-Cb-Hb moieties, according to the following equation [24] …”

“…The values of a, b and c were determined by best ®tting the d 2 -containing proteins to the dihedral angle values measured in the corresponding solution or solid state structure [24,25]. Finally, 58 constraints based on the nuclear relaxation enhancement were included after a critical evaluation [21]. The longitudinal relaxation rate enhancement of the I-th proton, R I , is proportional to the sixth power of the reciprocal distance between the nucleus and the n-th iron ion (r In ) [26]:…”