Mechanism for antibody catalysis of the oxidation of water by singlet dioxygen

Abstract

Wentworth et al. [Wentworth, P., Jones, L. H., Wentworth, A. D., Zhu, X. Y., Larsen, N. A., Wilson, I. A., Xu, X., Goddard, W. A., Janda, K. D., Eschenmoser, A. & Lerner, R. A. (2001) Science 293, 1806-1811] recently reported the surprising result that antibodies and T cell receptors efficiently catalyze the conversion of molecular singlet oxygen (1O2) plus water to hydrogen peroxide (HOOH). Recently, quantum mechanical calculations were used to delineate a plausible mechanism, involving reaction of 1O2 with two waters to form HOOOH (plus H2O), followed by formation of HOOOH dimer, which rearranges to form HOO-HOOO + H2O, which rearranges to form two HOOH plus 1O2 or 3O2. For a system with 18O H2O, this mechanism leads to a 2.2:1 ratio of 16O:18O in the product HOOH, in good agreement with the ratio 2.2:1 observed in isotope experiments by Wentworth et al. In this paper we use docking and molecular dynamics techniques (HierDock) to search various protein structures for sites that stabilize these products and intermediates predicted from quantum mechanical calculations. We find that the reaction intermediates for production of HOOH from 1O2 are stabilized at the interface of light and heavy chains of antibodies and T cell receptors. This inter Greek key domain interface structure is unique to antibodies and T cell receptors, but is not present in beta2-microglobulin, which does not show any stabilization in our docking studies. This result is consistent with the experimentally observed lack of HOOH production in this system. Our results provide a plausible mechanism for the reactions and provide an explanation of the specific structural character of antibodies responsible for this unexpected chemistry.

Figure 1

Gas phase structures (optimized using quantum mechanics; see ref. for various clusters and transition states). These structures were used in the docking studies.

Figure 2

(a) Clustering sites for docking of HOOOH dimers. All sites are located between the V_L and V_H interface. This shows regions I1 and I3 in front. Region I2 is opposite region I1 in the back. Inset shows where this region is relative to the overall Ig. Regions I1–I3 are in the IGKD unique to antibodies and TCR. (b) Clustering sites for docking of H₂O₂. Region P1 is situated within the β-barrel created by the V_H and V_L interface. P2 is located between the C_H and C_L interface. Regions P1–P2 are in the IGKD unique to antibodies and TCR.

Figure 3

(a) The purple dots indicate two regions of the Fab antibody fragment that bind strongly to the HOOOH dimer and that we conclude are plausible regions for the catalysis of ¹O₂ plus H₂O dimer to form HOOOH. These sites are at the interface of the V_H and V_L in a region containing well ordered crystallographic waters (shown with half bonds). These regions are in the IGKD unique to antibodies and TCR. Inset shows a schematic antibody structure with a yellow circle to indicate the region magnified. (b) The structure in a is rotated 90° about the horizontal axis to show the hydrophobic channel bounded by Gln-38 from V_L and Gln-39 from V_H. This forms a hydrogen bond network at the mouth of the barrel. Inset shows the barrel-like structure (containing two Greek keys) unique to antibodies that we suggest is critical to the catalysis.

Figure 4

Ordered water molecules found in the x-ray structure at the IGKD interface of the Fab antibody fragment. (a) A pentamer ring of H₂O molecules with each hydrogen bonded to two others. (b) An example water dimer where hydrogen from one water molecule is pointing toward the oxygen of the other molecule.

Figure 5

The geometric pathway for the sequence of reactions converting ¹O₂ water to HOOOH and then to HOOH. Here we assume that ¹O₂ enters the hydrophobic region near Xe1. At I1 (or I2) it can react with a water dimer (or trimer) to form HOOOH. The HOOOH may stay at I1 (or I2) but it may go to I3, which does not have crystallographic waters. This HOOOH may react directly with a second ¹O₂ or with the HOOOH from a previous reaction to form the HOOOH dimer—this may occur at I3. The HOOOH dimer can rearrange through a series of steps to form HOOH, which may go to sites P1 or P2 (there are no crystallographic waters at these points). Here the HOOH is positioned close to the region at which antigen may be bound (HOOOH may also go to this region). From here the HOOH (or HOOOH) might react directly with the part of a protein whose antigen is recognized by the antibody.