Multiple active site histidine protonation states in Acetobacter aceti N5-carboxyaminoimidazole ribonucleotide mutase detected by REDOR NMR

Abstract

Class I PurE (N5-carboxyaminoimidazole mutase) catalyzes a chemically unique mutase reaction. A working mechanistic hypothesis involves a histidine (His45 in Escherichia coli PurE) functioning as a general acid, but no evidence for multiple protonation states has been obtained. Solution NMR is a peerless tool for this task but has had limited application to enzymes, most of which are larger than its effective molecular size limit. Solid-state NMR is not subject to this limit. REDOR NMR studies of a 151 kDa complex of uniformly 15N-labeled Acetobacter aceti PurE (AaPurE) and the active site ligand [6-13C]citrate probed a single ionization equilibrium associated with the key histidine (AaPurE His59). In the AaPurE complex, the citrate central carboxylate C6 13C peak moves upfield, indicating diminution of negative charge, and broadens, indicating heterogeneity. Histidine 15N chemical shifts indicate His59 exists in approximately equimolar amounts of an Ndelta-unprotonated (pyridine-like) form and an Ndelta-protonated (pyrrole-like) form, each of which is approximately 4 A from citrate C6. The spectroscopic data are consistent with proton transfers involving His59 Ndelta that are invoked in the class I PurE mechanism.

Figure 1

Carbonyl-region ¹³C{¹⁵N} REDOR NMR spectra of the AaPurE•citrate complex after 2.56 ms of dipolar evolution (black) and 15.36 ms of dipolar evolution (red). The full-echo spectra (scaled to compensate for homogeneous decay) are shown at the bottom of the figure and the dephased echoes at the top. The peaks at 155 ppm and between 171 and 175 ppm arise from natural-abundance ¹³C in [U-¹⁵N]AaPurE and these are totally dephased (reduced to zero intensity) after 2.56 ms by directly bonded ¹⁵N. The remaining two peaks are assigned to ¹³C label in free (180 ppm) and bound (172 ppm) citrate. The latter undergoes about 60% dephasing after 15.36 ms consistent with a 3-Å ¹³C-¹⁵N intermolecular distance. Magic-angle spinning was at 6250 Hz.

Figure 2

¹⁵N{¹³C} REDOR NMR spectra of the AaPurE•citrate complex after 17.92 ms of dipolar evolution. Full-echo spectra (S₀) are shown at the bottom and middle of the figure and the REDOR difference (ΔS = S₀ − S, where S is the dephased-echo spectrum) at the top. The histidine nitrogen peaks at 150 and 220 ppm have much more dephasing (ΔS/S₀ expressed as percentages) than other sidechain peaks, consistent with proximity of the citrate ¹³C label to His59 (inset). The chemical-shift scale is relative to external solid ammonium sulfate. (To convert to the chemical shift scale more commonly used in protein NMR, which is referenced to the ¹⁵N frequency of liquid ammonia (32), add 24 ppm to the values given.) Magic-angle spinning was at 8000 Hz. Inset, a depiction of superimposed active sites A and B from the AaPurE•citrate complex crystal structure (PDB id 1u11) showing the two different citrate locations. Subunit A is depicted in ball-and-stick representations and subunit B is depicted in thick black lines. The subunit A ¹⁵N–¹³C distance is 3.34 Å and the subunit B ¹⁵N–¹³C distance is 3.53 Å. Structure superposition was performed using all backbone atoms in SwissPDB Viewer (19). The drawing was rendered with Molscript (20) and Raster3D (21).

Figure 3

Potential interactions between citrate and His59 in the AaPurE•citrate complex, with (right) or without (left) protonation. Atoms labeled C and N contain either ¹³C or ¹⁵N. Cit, citrate with a deprotonated central carboxylate; CitH, citrate with a protonated central carboxyl; Hid, N^δ-protonated His59; Hie, N^ε-protonated His59; Hip, fully protonated (imidazolium) form of His59. Both Hid and Hip would be consistent with an ¹⁵N^δ peak at 150 ppm. Hie gives an ¹⁵N^δ peak at 220 ppm. A single AaPurE•citrate complex is excluded by dephasing by nearby ¹³C of two separate ¹⁵N peaks assigned to different states of His59 N^δ. Crystallographically determined (PDB id 1u11) inter-atomic distances from His59 N^δ to the nearest citrate carboxylate O atom are 2.47 Å (subunit A) and 2.56 Å (subunit B). Since both dephased ¹⁵N peaks overlap with other His sidechain ¹⁵N peaks, there is no evidence for an unusual electronic environment associated with the "shared" hydrogen bonding interaction illustrated, where the proton is located near the center of the hydrogen bond.

Scheme 1

Possible mechanism for the reversible class I PurE-mediated conversion of N⁵-CAIR to CAIR.a ^a R5P is ribose 5'-phosphate.