15N-NMR characterization of His residues in and around the active site of FeSOD

Abstract

We have exploited (15)N-NMR to observe histidine (His) side chains in and around the active site of Fe-containing superoxide dismutase (FeSOD). In the oxidized state, we observe all the non-ligand His side chains and in the reduced state we can account for all the signals in the imidazole spectral region in terms of the non-ligand His', paramagnetically displaced signals from two backbone amides, and the side chain of glutamine 69 (Gln69). We also observe signals from the His' that ligate Fe(II). These confirm that neither the Q69H nor the Q69E mutation strongly affects the Fe(II) electronic structure, despite the 250 mV and >660 mV increases in E(m) they produce, respectively. In the Q69H mutant, we observe two new signals attributable to the His introduced into the active site in place of Gln69. One corresponds to a protonated N and the other is strongly paramagnetically shifted, to 500 ppm. The strong paramagnetic effects support the existence of an H-bond between His69 and the solvent molecule coordinated to Fe(II), as proposed based on crystallography. Based on previous information that His69 is neutral, we infer that the shifted N is not protonated. Therefore, we propose that this N represents a site of H-bond acceptance from coordinated solvent, representing a reversal of the polarity of this H-bond from that in WT (wild-type) FeSOD protein. We also present evidence that substrate analogs bind to Fe(II)SOD outside the Fe(II) coordination sphere, affecting Gln69 but without direct involvement of His30.

Figure 1

Diagram of the active site of FeSOD (left) and ribbon diagram of a FeSOD monomer in the context of the dimer (right). The active site is shown to include the ligands to Fe as well as His30, Tyr34 and Gln69 (counter clockwise from the left), based on the crystallographic coordinates of Lah et al.[9]. The residues introduced by mutation of Gln69 to His and Glu are shown as well, with the replacement residues shown as placed by overlays of each mutant active site with the WT site. The His69 of Q69H-FeSOD is depicted with green C and the Glu69 of Q69E-Fe²⁺SOD is depicted with yellow C atoms. The ribbon diagram of FeSOD shows the ligand histidines in CPK, non-ligand His’ in amber and the His introduced in the case of the Q69H mutant in green, for one monomer of the dimer. The other monomer is between the viewer and the page, and is shown as a partially transparent thin ribbon structure. Active site overlays optimized the superposition of all side-chain atoms of the amino acid residues 26, 30, 34, 73, 76 122, 156, 158, 160 and 161, but did not take into account residue 69, the Fe or the coordinated solvent. Overlays were performed using swisspdbviewer [58], the coordinates 1ZA5 and 2BKB of Yikilmaz et al,[33] and the coordinates 1ISA of Lah et al. [9]. Figures were generated using Molscript [59] and Raster 3D [60].

Figure 2

Dispersion of different protein functionalities over different regions of the 15N spectrum. In order to avoid invoking the ¹⁵N-nOe, the spectrum was collected without ¹H decoupling. Data were acquired at 10 °C over 0.25 s and 2 s recovery delay was allowed prior to the next 60 ° excitation pulse. An 80 ms Gaussian line broadening function was applied.

Figure 3

¹⁵N spectra of WT FeSOD, comparing the oxidized state (bottom) with the reduced state (top two). For the reduced state, comparison of the top spectrum collected without ¹H decouping, and the middle spectrum collected with ¹H decoupling, identifies N sites with a slowly-exchanging ¹H bound, as split in the top spectrum (orange vertical lines). A blue brace identifies a region including four overlapping resonances in WT-Fe^IISOD, ‘A’ identifies resonances attributed to backbone amides, ‘Q’ identifies the broad resonance of Gln69 and green vertical lines identify resonances that appear common to the reduced and oxidized states. All spectra were collected at 10 °C. A 45° excitation pulse and 100 ms acquisition were used with 2 s relaxation delays between scans.

Figure 4

Chemical shifts of strongly-shifted and relaxed resonances of WT- (blue) and Q69E0Fe^IISOD (red) as functions of 1000*1/T. (Q69H-Fe^IISOD did not tolerate temperatures above 10°C for the long intervals needed to collect ¹⁵N spectra.) Data were collected at each of 10, 25 and 40 °C. 80,000 scans using a 30 ° excitation pulse (to cover a broad spectral width), 60 ms of acquisition and a 1 s relaxation delay between scans. 5 ms Gaussian line broadening was applied.

Figure 5

Comparison of the imidazole-region of the ¹⁵N spectrum of WT-Fe^IISOD with those of Q69E-Fe^IISOD (top two spectra) and Q69H-Fe^IISOD (lower two spectra). A 90° excitation pulse and 250 ms acquisition were used with 5 s relaxation delays between scans.

Figure 6

Comparison of the down-field region of ¹⁵N spectra of WT-, Q69E- and Q69H-Fe^IISOD. A 90° excitation pulse and 250 ms acquisition were used with 5 s relaxation delays between scans. 5 ms Gaussian line broadening was applied.

Figure 7

Effect of addition of the substrate analog F⁻ to WT-Fe^IISOD. WT-Fe^IISOD was in 10 mM morpholinoethanesulfonate (MES) buffer at pH 6.0 with 10 mM NaCl with (top) or without (bottom) addition of 100 mM KF. The K_d for F⁻ binding to Fe²⁺SOD is 40 mM (F⁻ inhibits FeSOD with a K_I of 19 mM but binds to oxidized Fe³⁺SOD with a K_d of 7 mM) A 30° excitation pulse and 60 ms acquisition were used with 1 s relaxation delays between scans. The control spectrum (no F⁻) displays two signals that are attributed to damaged protein in this older sample, near 175 and 215 ppm. These are not present in fresher material, see for example Figure 3.