Catalytic cycle of human glutathione reductase near 1 A resolution

Abstract

Efficient enzyme catalysis depends on exquisite details of structure beyond those resolvable in typical medium- and high-resolution crystallographic analyses. Here we report synchrotron-based cryocrystallographic studies of natural substrate complexes of the flavoenzyme human glutathione reductase (GR) at nominal resolutions between 1.1 and 0.95 A that reveal new aspects of its mechanism. Compression in the active site causes overlapping van der Waals radii and distortion in the nicotinamide ring of the NADPH substrate, which enhances catalysis via stereoelectronic effects. The bound NADPH and redox-active disulfide are positioned optimally on opposite sides of the flavin for a 1,2-addition across a flavin double bond. The new structures extend earlier observations to reveal that the redox-active disulfide loop in GR is an extreme case of sequential peptide bonds systematically deviating from planarity--a net deviation of 53 degrees across five residues. But this apparent strain is not a factor in catalysis, as it is present in both oxidized and reduced structures. Intriguingly, the flavin bond lengths in oxidized GR are intermediate between those expected for oxidized and reduced flavin, but we present evidence that this may not be due to the protein environment but instead due to partial synchrotron reduction of the flavin by the synchrotron beam. Finally, of more general relevance, we present evidence that the structures of synchrotron-reduced disulfide bonds cannot generally be used as reliable models for naturally reduced disulfide bonds.

Figure 1

Catalytic cycle of glutathione reductase. The native state with no substrate bound is not part of the cycle but merely forms an entrance point. Dotted lines indicate charge-transfer complexes between NADPH, FAD, and the sulfur of Cys63. The substrate and product binding and dissociation may occur with different timing than that shown. The four crystal structures reported here provide information about the catalytic intermediates as follows: 1 is GR_Native; 2 are derived from GR_Native and GR_NADPH; 3 is derived from GR_NADPH and GR_GSSG/NADP; 4 is GR_NADPH; 5 is derived from GR_GSSG/NADP and GR_NADPH; 6 is derived from GR_GSH and GR_NADPH; 7 is derived from GR_Native and GR_NADPH, with GR_GSSG/NADP and GR_GSH providing an idea for the GSH_I binding site. Created in Inkscape.

Figure 2

Atomic-resolution electron density for the active-site cofactors. a) The nicotinamide ring of NADPH at 1.0 Å resolution (contour level 3.2*ρ_rms) from GR_NADPH, with carbons (cyan), nitrogens (blue) and oxygens (red) having distinct electron density levels. The C4 atom, which transfers a hydride to FAD, is at the bottom. Pyramidalization of atom N1, at the top of the ring, is visible. A slight twist in the carboxamide relative to the ring is also visible. b) The flavin ring system of FAD at 0.95 Å resolution (contour level 3.8*ρ_rms) from GR_Native, with coloring as in (a) except carbons are green. The N5 atom, where FAD receives electrons from NADPH, is at the bottom center. A small twist in the flavin is evident.

Figure 2

Atomic-resolution electron density for the active-site cofactors. a) The nicotinamide ring of NADPH at 1.0 Å resolution (contour level 3.2*ρ_rms) from GR_NADPH, with carbons (cyan), nitrogens (blue) and oxygens (red) having distinct electron density levels. The C4 atom, which transfers a hydride to FAD, is at the bottom. Pyramidalization of atom N1, at the top of the ring, is visible. A slight twist in the carboxamide relative to the ring is also visible. b) The flavin ring system of FAD at 0.95 Å resolution (contour level 3.8*ρ_rms) from GR_Native, with coloring as in (a) except carbons are green. The N5 atom, where FAD receives electrons from NADPH, is at the bottom center. A small twist in the flavin is evident.

Figure 3

Disulfide bonds reduced by radiation at cryotemperatures are different from those reduced chemically. The GR_Native (green carbons) structure shows both the native disulfide and an alternate conformation for Cys58 due to radiation damage at cryotemperatures. GR_NADPH (cyan carbons) shows the structure resulting from chemical reduction at room temperature. The overlay shows a clear difference in the backbone relaxation of Cys58 that depends upon the mode of reduction.

Figure 4

Peptide non-planarity in the active-site disulfide loop. a) Stereoview of the disulfide loop with standard hydrogen bonds (green dotted lines) and unusually long “hydrogen bonds” (red dotted lines) shown. b) Views down each peptide bond in the loop in GR_Native visually reveal the magnitude of omega deviations from planarity, which are 4°, 13°, 7°, 10°, 5°, and 11° for residues 58–63. c) A plot of smoothed (N=5) omega deviations from planarity shows the disulfide loop (residues 59–63 in particular) as the most consistently non-planar region in GR. Omega deviations in this loop are similar in all four structures. d) Histogram of pentapeptide stretches in atomic-resolution structures with deviations from planarity (see Methods). The level of nonplanarity of this GR loop (ranging from 46° to 53° in the four GR structures) is unusual, with only two other examples of similarly deviating loops (see Results & Discussion).

Figure 4

Peptide non-planarity in the active-site disulfide loop. a) Stereoview of the disulfide loop with standard hydrogen bonds (green dotted lines) and unusually long “hydrogen bonds” (red dotted lines) shown. b) Views down each peptide bond in the loop in GR_Native visually reveal the magnitude of omega deviations from planarity, which are 4°, 13°, 7°, 10°, 5°, and 11° for residues 58–63. c) A plot of smoothed (N=5) omega deviations from planarity shows the disulfide loop (residues 59–63 in particular) as the most consistently non-planar region in GR. Omega deviations in this loop are similar in all four structures. d) Histogram of pentapeptide stretches in atomic-resolution structures with deviations from planarity (see Methods). The level of nonplanarity of this GR loop (ranging from 46° to 53° in the four GR structures) is unusual, with only two other examples of similarly deviating loops (see Results & Discussion).

Figure 4

Peptide non-planarity in the active-site disulfide loop. a) Stereoview of the disulfide loop with standard hydrogen bonds (green dotted lines) and unusually long “hydrogen bonds” (red dotted lines) shown. b) Views down each peptide bond in the loop in GR_Native visually reveal the magnitude of omega deviations from planarity, which are 4°, 13°, 7°, 10°, 5°, and 11° for residues 58–63. c) A plot of smoothed (N=5) omega deviations from planarity shows the disulfide loop (residues 59–63 in particular) as the most consistently non-planar region in GR. Omega deviations in this loop are similar in all four structures. d) Histogram of pentapeptide stretches in atomic-resolution structures with deviations from planarity (see Methods). The level of nonplanarity of this GR loop (ranging from 46° to 53° in the four GR structures) is unusual, with only two other examples of similarly deviating loops (see Results & Discussion).

Figure 4

Peptide non-planarity in the active-site disulfide loop. a) Stereoview of the disulfide loop with standard hydrogen bonds (green dotted lines) and unusually long “hydrogen bonds” (red dotted lines) shown. b) Views down each peptide bond in the loop in GR_Native visually reveal the magnitude of omega deviations from planarity, which are 4°, 13°, 7°, 10°, 5°, and 11° for residues 58–63. c) A plot of smoothed (N=5) omega deviations from planarity shows the disulfide loop (residues 59–63 in particular) as the most consistently non-planar region in GR. Omega deviations in this loop are similar in all four structures. d) Histogram of pentapeptide stretches in atomic-resolution structures with deviations from planarity (see Methods). The level of nonplanarity of this GR loop (ranging from 46° to 53° in the four GR structures) is unusual, with only two other examples of similarly deviating loops (see Results & Discussion).

Figure 5

Nicotinamide binding tightens the active site. a) Anisotropic mobility is shown as thermal ellipsoids for residues as seen in the active site of the GR_GSSG/NADP complex (carbons violet). b) The same view for the GR_NADPH complex (carbons cyan). In the NADPH complex, the motion is much lower and more isotropic.

Figure 5

Nicotinamide binding tightens the active site. a) Anisotropic mobility is shown as thermal ellipsoids for residues as seen in the active site of the GR_GSSG/NADP complex (carbons violet). b) The same view for the GR_NADPH complex (carbons cyan). In the NADPH complex, the motion is much lower and more isotropic.

Figure 6

Steric compression in nicotinamide-flavin interaction. Side view of overlaid active site centered on flavin, showing GR_Native (green), GR_GSH (magenta) and GR_NADPH (cyan). In GR_NADPH, NADPH binding above the flavin pushes it down into Cys63, and in GR_GSH, GSH and the Cys63 thiolate below the flavin push it up. GR_GSSG/NADP is not shown because its atoms are in the same positions as in GR_Native. To conserve space the flavin-nicotinamide and the flavin-Cys63 separations are not to scale. Figure 8 shows these distances to scale.

Figure 7

The nicotinamide distortion and ribose conformation favor catalysis. a) The schematic shows the planes of the nicotinamide and flavin (solid black lines). The hypothesized partial boat is shown as a solid red line. Pyramidalization at the nicotinamide N1 places the lone pair on the flavin side where it (i) entropically favors the productive boat conformation to form and (ii) repels the hydride to be transferred (dashed red line). b) The ribose conformation relative to the nicotinamide stabilizes the electron-deficient NADP⁺ ring orbitals via hyperconjugative electron donation from the ribose. The glycosidic C-O bond position parallel to the nicotinamide ring also favors NADP⁺ over NADPH (see Results & Discussion).

Figure 7

The nicotinamide distortion and ribose conformation favor catalysis. a) The schematic shows the planes of the nicotinamide and flavin (solid black lines). The hypothesized partial boat is shown as a solid red line. Pyramidalization at the nicotinamide N1 places the lone pair on the flavin side where it (i) entropically favors the productive boat conformation to form and (ii) repels the hydride to be transferred (dashed red line). b) The ribose conformation relative to the nicotinamide stabilizes the electron-deficient NADP⁺ ring orbitals via hyperconjugative electron donation from the ribose. The glycosidic C-O bond position parallel to the nicotinamide ring also favors NADP⁺ over NADPH (see Results & Discussion).

Figure 8

Stereoelectronic control in nicotinamide-flavin interaction. (a) A side view with the flavin N5-C4a bond in the plane of the paper and (b) a view down the flavin N5-C4a bond together show the optimal geometry for concerted 1–2 addition across the double bond. Compression in the form of shorter than van der Waals interactions is also shown in (a).

Figure 9

Data quality as a function of resolution. Observed data quality is indicated by R_meas values and plotted as a function of resolution⁻¹. The four structures are colored as indicated in the key. These data were used to determine where to set the high-resolution cutoff (see Methods).