Fig. 3: The suggested mechanism of Gln143 → WAT1 proton transfer utilizing bonding orbitals and optimal resonance forms calculated from chemist’s localized property-optimized orbital (CLPO) analysis of the neutron structures.

Orange and purple contours indicate the positive and negative orbital wave functions, respectively. Curved arrows represent electron pushing. a, b The reduction of Mn³⁺ to Mn²⁺ instigates a strong covalent need for the exposed lone pair (denoted LP) of ^–OH(WAT1). This is chemically remediated by the acquisition of a proton (green atom) from the proximal Gln143 amide. The dominant resonance structure of the amide anion is with a N^ε2–C^ε1 double bond as a result of N^ε2 LP delocalization from a. c Despite proton donation to WAT1, Gln143 still demonstrates covalent character with the proton (green atom). The new N^ε2 LP participates in electron density transfer to the σ^*-antibonding orbital of O–H(WAT1). Donor acceptor orbital analysis calculates a stabilizing energy of 1.4 kcal mol⁻¹ for this hyperconjugated interaction. The inset in the lower-right corner illustrates individual orbital representations. d Due to the hyperconjugation illustrated in c, the hydrogen bond between N^ε2(Gln143) and O–H(WAT1) has partial σ-bonding character contributing to and increasing the strength of the hydrogen bond characteristic of short-strong hydrogen bonds (SSHBs). CLPO calculations suggest the proton is covalently shared between WAT1 and Gln143, with percentages of 64 and 36%, respectively. e, f The increased electronegative character of O^ε1 electrostatically polarizes Trp123. This is achieved through the stabilizing delocalization of the N^ε1(Trp123) LP into the conjugated ring. The major stabilizing interaction decreases energy by 13.52 kcal mol⁻¹ and is the donation of electron density from the N^ε1(Trp123) LP into the adjacent C^ε2–C^δ2 π ^*-antibonding orbital. The polarization of Trp123 allows an SSHB between Gln143 and Trp denoted by the green dashes. The hyperconjugation and polarization of Trp123 are thought to contribute to the stability of the amide anion.