Low-barrier hydrogen bond inside a protein seen directly with neutrons… tracing what makes a proton shared

• A US team combined neutron crystallography, atomic resolution X-ray crystallography and quantum mechanical simulation to directly observe a low-barrier hydrogen bond (LBHB) in the bacterial protein YajL.
• The homologous human protein DJ-1 carries a conventional hydrogen bond at the same position, but mutating a single residue outside the bond (I21T) increased proton delocalization and gave the interaction LBHB-like character.
• The work shows that an extended hydrogen-bond network inside a protein can modulate the proton transfer barrier, while leaving the biological function of the LBHB itself an open question.

[Quantum Biology Society]

Hydrogen bonds are the interactions that shape protein structure and enzyme activity. In most of them the proton stays firmly attached to its donor atom, but when the acid dissociation constants (pKa) of donor and acceptor become comparable and the two atoms come close together, a low-barrier hydrogen bond (LBHB) can form, in which the proton moves freely between the two atoms. The proposal that LBHBs boost enzyme catalysis has been argued over for more than thirty years, and testing it has been hampered by how hard it is to pin down experimentally the position of a light hydrogen atom.

A collaboration between the University of Nebraska-Lincoln, Oak Ridge National Laboratory (ORNL) and Wayne State University has now brought decisive experimental evidence to that debate, reporting in the journal Biochemistry in February 2026. The team worked with human DJ-1, a protein linked to Parkinson's disease, and its Escherichia coli homologue YajL, using isotopic substitution together with three mutually independent techniques to track exactly where the proton sits.

■ Cross-validating the LBHB with multiple techniques

To get past the limitation of X-ray crystallography, which reports electrons rather than nuclei, the researchers turned to neutron crystallography, where neutrons scatter from atomic nuclei and the position of a hydrogen (deuterium) atom can be located directly.

• Neutron diffraction: in the 1.79 Å resolution neutron map of YajL, the deuteron sits almost equidistant from the two oxygen atoms of Glu14 and Asp23, the signature of an LBHB, and it does so in both protomers. In the 2.15 Å map of DJ-1, by contrast, the deuteron is displaced toward Asp24, marking a conventional hydrogen bond.
• Atomic resolution X-ray bond length analysis: protonation was judged indirectly from the difference between C-O and C=O bond lengths, and the verdict was consistent with the neutron result.
• QM/MM simulation: in quantum mechanics/molecular mechanics Born-Oppenheimer molecular dynamics (QM/MM-BOMD) and the accompanying free energy calculations, the proton transfer barrier in YajL was only about 1.1 kcal/mol and the proton ranged widely between the two oxygen atoms, while DJ-1 showed a barrier of more than 3 kcal/mol that held the proton on one side.

■ Microenvironmental differences and the control of proton delocalization

Despite their structural similarity, the two proteins behave differently at this bond. They share 41% sequence identity and superimpose to within 1.1 Å Cα RMSD, yet one has an LBHB and the other does not. The team predicted that nearby residues, which take no direct part in the bond itself, fine-tune the pKa values through the surrounding hydrogen-bond network.

They tested this by replacing Ile21 of DJ-1 with the polar threonine found at the equivalent position in YajL. In the resulting I21T variant the proton moved out toward the midpoint between the two oxygen atoms, showing delocalization approaching that of an LBHB, and the barrier to reaching the shared position fell to 1.15 kcal/mol, close to the value in YajL, although the proton remained somewhat biased toward the Asp donor. The result indicates that the proton transfer barrier is set not by a local interaction between two atoms alone but by the wider protein microenvironment surrounding the hydrogen bond.

■ Significance and open questions

The chief value of this work is that it uses demanding neutron experiments to validate the reliability of X-ray bond length analysis, and that it establishes an experimental handle for tuning the degree of proton sharing through a remote mutation alone.

The authors were careful not to declare a biological role for the phenomenon. They state that the specific biological function of the LBHB found in YajL remains unknown, and that any connection to catalysis at the active site is still at the level of a hypothesis. What the work offers is a set of precise measurement and control tools with which the thirty-year argument over the functional role of LBHBs can finally be put to rigorous experimental and computational test, which should open a new chapter in the study of enzyme dynamics.

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https://pubs.acs.org/doi/10.1021/acs.biochem.5c00762