The vibrational theory of olfaction assumes that electron transfer occurs across odorants at the active sites of odorant receptors (ORs), serving as a sensitive measure of odorant vibrational frequencies, ultimately leading to olfactory perception. A previous study reported that human subjects differentiated hydrogen/deuterium isotopomers (isomers with isotopic atoms) of the musk compound cyclopentadecanone as evidence supporting the theory. Here, we find no evidence for such differentiation at the molecular level. In fact, we find that the human musk-recognizing receptor, OR5AN1, identified using a heterologous OR expression system and robustly responding to cyclopentadecanone and muscone, fails to distinguish isotopomers of these compounds in vitro. Furthermore, the mouse (methylthio)methanethiol-recognizing receptor, MOR244-3, as well as other selected human and mouse ORs, responded similarly to normal, deuterated, and ¹³C isotopomers of their respective ligands, paralleling our results with the musk receptor OR5AN1. These findings suggest that the proposed vibration theory does not apply to the human musk receptor OR5AN1, mouse thiol receptor MOR244-3, or other ORs examined. Also, contrary to the vibration theory predictions, muscone-d₃₀ lacks the 1,380- to 1,550-cm⁻¹ IR bands claimed to be essential for musk odor. Furthermore, our theoretical analysis shows that the proposed electron transfer mechanism of the vibrational frequencies of odorants could be easily suppressed by quantum effects of nonodorant molecular vibrational modes. These and other concerns about electron transfer at ORs, together with our extensive experimental data, argue against the plausibility of the vibration theory. Significance The vibrational theory of olfaction posits detection of odorants through their vibrational frequencies rather than solely through “hand-in-glove” substrate/enzyme-like odorant–odorant receptor (OR) interactions. To test the theory, we compare responses of different human and mouse ORs toward deuterated and undeuterated isotopomers (isotopic atom isomers) of receptor-responsive odorants because isotopomers should differ in their molecular vibrational frequencies. However, no differences in receptor response are seen with any tested labeled/unlabeled odorant/receptor pairs. Because published behavioral studies have shown that humans can distinguish isotopomers, perireceptor events or impurities, rather than receptor-level vibrational effects, are suggested. Because theoretical aspects of the vibration theory are also found wanting, the vibration theory is deemed implausible in the absence of compelling receptor-level experimental evidence to the contrary.

Understanding olfaction at the molecular level is challenging due to the lack of crystallographic models of odorant receptors (ORs). To better understand the molecular mechanism of OR activation, we focused on chiral (R)-muscone and other musk-smelling odorants due to their great importance and widespread use in perfumery and traditional medicine, as well as environmental concerns associated with bioaccumulation of musks with estrogenic/antiestrogenic properties. We experimentally and computationally examined the activation of human receptors OR5AN1 and OR1A1, recently identified as specifically responding to musk compounds. OR5AN1 responds at nanomolar concentrations to musk ketone and robustly to macrocyclic sulfoxides and fluorine-substituted macrocyclic ketones; OR1A1 responds only to nitromusks. Structural models of OR5AN1 and OR1A1 based on quantum mechanics/molecular mechanics (QM/MM) hybrid methods were validated through direct comparisons with activation profiles from site-directed mutagenesis experiments and analysis of binding energies for 35 musk-related odorants. The experimentally found chiral selectivity of OR5AN1 to (R)- over (S)-muscone was also computationally confirmed for muscone and fluorinated (R)-muscone analogs. Structural models show that OR5AN1, highly responsive to nitromusks over macrocyclic musks, stabilizes odorants by hydrogen bonding to Tyr260 of transmembrane α-helix 6 and hydrophobic interactions with surrounding aromatic residues Phe105, Phe194, and Phe207. The binding of OR1A1 to nitromusks is stabilized by hydrogen bonding to Tyr258 along with hydrophobic interactions with surrounding aromatic residues Tyr251 and Phe206. Hydrophobic/nonpolar and hydrogen bonding interactions contribute, respectively, 77% and 13% to the odorant binding affinities, as shown by an atom-based quantitative structure–activity relationship model. Significance While natural musk has been used for 2,000 years in perfumery, and in traditional medicine for its cardioprotective effects, its mode of activating odorant receptors (ORs) is unknown. ORs, G protein-coupled receptors (GPCRs), which constitute 40% of all pharmacophore receptors, are also expressed in nonolfactory tissues. Understanding the activation of ORs at the molecular level is challenging due to lack of crystallographic models. By combining site-directed mutagenesis with computational studies of human musk ORs involving 35 chiral and achiral muscone analogues, we propose structural models, including binding site prediction and responsible amino acid residues identification. Our studies of musk-responsive ORs should assist the study of the pharmacological effects of musks involving non-OR GPCRs.