Abstract
Fundamental vibrations of the chromophore in the membrane protein bacteriorhodopsin (BR), a protonated Schiff base retinal, have been studied for decades, both by resonance Raman and by infrared (IR) difference spectroscopy. Such studies started comparing vibrational changes between the initial BR state (all-trans retinal) and the K intermediate (13-cis retinal), being later extended to the rest of intermediates. They contributed to our understanding of the proton-pumping mechanism of BR by exploiting the sensitivity of fundamental vibrational transitions of the retinal to its conformation. Here, we report on new bands in the 2,500 to 1,800 cm−1 region of the K-BR difference FT-IR spectrum. We show that the bands between 2,500 and 2,300 cm−1 originate from overtone and combination transitions from C-C stretches of the retinal. We assigned bands below 2,300 cm−1 to the combination of retinal C-C stretches with methyl rocks and with hydrogen-out-of-plane vibrations. Remarkably, experimental C-C overtone bands appeared at roughly twice the wavenumber of their fundamentals, with anharmonic mechanical constants ≤3.5 cm−1, and in some cases of ∼1 cm−1. Comparison of combination and fundamental bands indicates that most of the mechanical coupling constants are also very small. Despite the mechanical quasi-harmonicity of the C-C stretches, the area of their overtone bands was only ∼50 to ∼100 times smaller than of their fundamental bands. We concluded that electrical anharmonicity, the second mechanism giving intensity to overtone bands, must be particularly high for the retinal C-C stretches. We corroborated the assignments of negative bands in the K-BR difference FT-IR spectrum by ab initio anharmonic vibrational calculations of all-trans retinal in BR using a quantum-mechanics/molecular mechanics approach, reproducing reasonably well the small experimental anharmonic and coupling mechanical constants. Yet, and in spite accounting for both mechanical and electrical anharmonicities, the intensity of overtone C-C transitions was underestimated by a factor of 4–20, indicating room for improvement in state-of-the-art anharmonic vibrational calculations. The relatively intense overtone and combination bands of the retinal might open the possibility to detect retinal conformational changes too subtle to significantly affect fundamental transitions but leaving a footprint in overtone and combination transitions.
Highlights
The vibrations of atoms within molecules are deeply connected with the geometry and force of their chemical bonds
In the K intermediate most of the structural changes are restricted to the chromophore and its vicinity (Neutze et al, 2002; Wickstrand et al, 2019), and, most of the resolved bands in the K-BR spectrum come from vibrational modes located at the retinal molecule (Rothschild et al, 1984)
Bands in the mid-IR region of proteins are, by default, often assumed to corresponds to fundamental transitions from molecular vibrations. While this assumption is generally correct, in this work we have shown that the light-induced FT-IR difference spectrum of bacteriorhodopsin contains bands in the mid-IR region coming not from fundamental but from overtone and combination transitions
Summary
The vibrations of atoms within molecules are deeply connected with the geometry and force of their chemical bonds. The interpretation of changes in positions, intensities and/or widths of bands in IR difference spectra of proteins is often based on simple rules of thumb about the effects of H-bonding, polarity and vibrational coupling on vibrational properties (Barth and Zscherp, 2002; Lorenz-Fonfria, 2020), rarely specific enough to provide quantitative atomistic predictions In this context, vibrational calculations are gaining popularity as a tool to guide the interpretation of experimental spectra in atomic terms, resolving ambiguities about protonation states and H-bonds of groups (Domratcheva et al, 2016; Nakamura and Noguchi, 2016, 2017; Peuker et al, 2016), or tautomers (Domratcheva et al, 2016), complementing ambiguous or missing information in X-ray crystallographic structures of proteins. The utility of vibrational computations to interpret experimental IR spectra from biomolecules is still limited by our ability to accurately calculate IR spectra from input molecular structures
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