Implausibility of the vibrational theory of olfaction

Abstract

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 (13)C 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.

Keywords: cyclopentadecanone; electron transfer; isotopomers; muscone; olfaction.

Fig. 1.

(A) Preparation of deuterated 1–3. Deuterium could be selectively introduced, or selectively removed, adjacent to the carbonyl group using D₂O/K₂CO₃ or H₂O/K₂CO₃, respectively, at 130 °C; global replacement of all hydrogens could be achieved with Rh/C in D₂O at 150 °C. Repetition led to more complete deuteration as well as reduction of 1 to 3 and 2; oxidation of 2 gave 1 with ∼98% deuteration. Chromatography of deuterated 1 with freshly distilled pentane followed by repeated recrystallization from methanol/water to constant melting point gave samples showing no new peaks in their ¹H NMR spectra, other than very weak peaks corresponding to those peaks seen in undeuterated 1. (B) Deuterated (97%) muscone 4 was prepared via alcohol 5 as above. (C) 8-d₅ and 2,4,5,7-tetrathiaoctane-d_10, (9-d₁₀; 98% deuterium) were prepared as shown. Details of these syntheses are provided in SI Appendix.

Fig. 2.

Superimposed IR spectra of 4-d₃₀ (red trace) and undeuterated muscone (4; black trace) showing that 4-d₃₀ is devoid of IR absorption in the 1,380- to 1,550-cm⁻¹ region.

Fig. 3.

Dose–response curves of OR5AN1 to isotopomers of cyclopentadecanone (1) and muscone (4) (A) and MOR244-3 to isotopomers of MTMT (8), bis(methylthiomethyl) disulfide (9), and dimethyl sulfide (10) (B). Best-fit logEC₅₀ values of the curves are shown alongside the graph legends (placed below the graphs). Scatter plots with 95% confidence interval logEC₅₀ values and indicating statistical significances between the logEC₅₀ values among isotopomers are also shown below the corresponding graphs. In B, 30 μM of copper was added upon odorant stimulation. “SHAM” indicates nondeuterated cyclopentadecanone subjected to the same chemical synthetic procedures as the deuterated samples without D₂O addition. NS, not significant. For all dose–response curve graphs, the chemical structures of the respective compounds are shown within the graphs and normalized responses are shown as mean ± SEM (n = 3).

Fig. 4.

Dose–response curves of various mouse receptors to isotopomers of acetophenone (6) (A) and benzaldehyde (7) (B). Best-fit logEC₅₀ values of the curves are shown alongside the graph legends (placed below the graphs). Scatter plots with 95% confidence interval logEC₅₀ values and indicating statistical significances between the logEC₅₀ values among isotopomers are also shown below the corresponding graphs.

Fig. 5.

Natural logarithms of the ratios of τ₀/τ₁ vs. −ΔG⁰/λ (negative Gibbs free energy of reaction in the unit of reorganization energy) corresponding to the results in SI Appendix, Figs. S1.1–S1.3. The term “Classical” refers to the classical limit of Ohmic bath, SI Appendix, Eq. 18, with parameters for SI Appendix, Fig. S1.1. The term “Quantum-1” refers to the quantum regime of the Ohmic bath with parameters for SI Appendix, Fig. S1.2. The term “Quantum-2” refers to the case of classical Ohmic bath plus one quantum mode in the bath with parameters for SI Appendix, Fig. S1.3. Each column represents a different value of ħω_O/λ, where ω_O is the angular frequency of the odorant oscillator and λ is the reorganization energy of the protein environments. S_O = 0.01 (Upper) and S_O = 0.05 (Lower), where S_O is the Huang–Rhys factor for the odorant oscillator.