Low barrier hydrogen bonds (LBHBs) are H-bonds where the barrier to proton transfer from the donor to the acceptor atom is comparable to the quantum mechanical zero-point energy of the interaction, resulting in proton delocalization. Facile proton transfer makes LBHBs effective mechanisms for facilitating acid-base catalysis in certain enzymes, creating favored pathways of allosteric communication between distant sites during catalysis, and imparting a high degree of selectivity in binding certain substrates. How the microenvironment of H-bonded residues affects the degree of proton delocalization in a candidate LBHB is not fully understood. We use two homologous proteins, human DJ-1 and E. coli YajL, as a paired model system to investigate microenvironmental effects on LBHB formation. Neutron diffraction demonstrates that YajL has an Asp- Glu LBHB while the analogous residues in DJ-1 form a conventional H-bond, validating bond length analysis of atomic resolution X-ray crystal structures. We explored the influence of the microenvironment on LBHB character by using large quantum mechanics-molecular mechanics (QM/MM) simulations where the QM region spanned several residues. These simulations support the neutron and X-ray crystallography results, demonstrating that surprisingly distant H-bonding residues can influence the degree of proton delocalization in candidate LBHBs. With the greater accessibility of QM/MM methods, combining simulation and structurally guided sequence analysis may permit the engineering of H-bonds with desired degrees of proton delocalization.