Strong ionic hydrogen bonding causes a spectral isotope effect in photoactive yellow protein

Abstract

Standard hydrogen bonds are of great importance for protein structure and function. Ionic hydrogen bonds often are significantly stronger than standard hydrogen bonds and exhibit unique properties, but their role in proteins is not well understood. We report that hydrogen/deuterium exchange causes a redshift in the visible absorbance spectrum of photoactive yellow protein (PYP). We expand the range of interpretable isotope effects by assigning this spectral isotope effect (SIE) to a functionally important hydrogen bond at the active site of PYP. The inverted sign and extent of this SIE is explained by the ionic nature and strength of this hydrogen bond. These results show the relevance of ionic hydrogen bonding for protein active sites, and reveal that the inverted SIE is a novel, to our knowledge, tool to probe ionic hydrogen bonds. Our results support a classification of hydrogen bonds that distinguishes the properties of ionic hydrogen bonds from those of both standard and low barrier hydrogen bonds, and show how this classification helps resolve a recent debate regarding active site hydrogen bonding in PYP.

Figure 1

Anionic hydrogen bonding of pCA at the active site of PYP. A three-dimensional depiction of hydrogen bonding of the anionic pCA at the active site of wtPYP based on its crystal structure (PDB ID 1NWZ) (A). A two-dimensional depiction of hydrogen bonding of the pCA with residues 42 and 46, including hydrogen bonding lengths based on the crystal structures of wtPYP (B) and E46Q PYP (C) (PDB IDs: 1NWZ and 1OTA). To see this figure in color, go online.

Figure 2

Spectral isotope effects in PYP. The UV/vis absorbance spectra of the PYPs from S. ruber (top two curves) and H. halophila (bottom two curves) and the E46Q mutant of Sr PYP (middle two curves) in H₂O (black) and D₂O (red). The shifts in the chromophore absorbance bands upon dissolving the proteins in D₂O are indicated. To see this figure in color, go online.

Figure 3

Effects of H/D exchange on active site hydrogen bonding strength and absorbance maximum in PYP. The estimated weakening of the hydrogen bond between residue 46 and the chromophore upon H/D exchange, and the resulting experimentally detected redshift in absorbance maximum are depicted for the PYP from Salinibacter ruber and its E46Q mutant.

Figure 4

Distinction between three types of hydrogen bonds. The distance between heteroatoms in the hydrogen bond donor-acceptor pair (A) define the equilibrium length and strength of hydrogen bonds, whereas the position of the proton along this hydrogen bond (B) determines the potential energy for proton transfer between the hydrogen bond donor and acceptor. These two reaction coordinates lead to a distinction between normal hydrogen bonds (NHB) with a length of 3.00 (±0.20) Å, ionic hydrogen bond (IHB) with a length of 2.72 (±0.10) Å, and low-barrier hydrogen bonds (LBHB) with a length of 2.46 (±0.03) Å. The depicted potential energy curves are intended to reflect the qualitative differences between the three types of hydrogen bonds, while depicting physically reasonable values. Quantitative information on the shapes of these energy landscapes were obtained from (52–54). Although ionic hydrogen bonds and LBHBs are both considerably stronger than standard hydrogen bonds, the values for their strength are less well defined and likely to vary considerably depending on the details of the chemical nature of the donor-acceptor pair and their physical environment. Likewise, the exact values for the barriers and energy differences for proton transfer in panel B are intended to illustrate the differences between the three types of hydrogen bonds; the absolute energies of the right wells and the vibrational levels are for illustrative purposes. The depicted values were chosen to reflect hydrogen bonds involving O (and N) heteroatoms. Normal hydrogen bonds occur abundantly in proteins; ionic hydrogen bonds are found at some active sites and occur often on the surface of protein. LBHBs are rare and often are present at the active site of the protein.