Retinal Vibrations in Bacteriorhodopsin are Mechanically Harmonic but Electrically Anharmonic: Evidence From Overtone and Combination Bands

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.

Characterisation of the redox centers of ethylbenzene dehydrogenase

Ethylbenzene dehydrogenase (EbDH), the initial enzyme of anaerobic ethylbenzene degradation from the beta-proteobacterium Aromatoleumaromaticum, is a soluble periplasmic molybdenum enzyme consisting of three subunits. It contains a Mo-bis-molybdopterin guanine dinucleotide (Mo-bis-MGD) cofactor and an 4Fe–4S cluster (FS0) in the α-subunit, three 4Fe–4S clusters (FS1 to FS3) and a 3Fe–4S cluster (FS4) in the β-subunit and a heme b cofactor in the γ-subunit. Ethylbenzene is hydroxylated by a water molecule in an oxygen-independent manner at the Mo-bis-MGD cofactor, which is reduced from the MoVI to the MoIV state in two subsequent one-electron steps. The electrons are then transferred via the Fe–S clusters to the heme b cofactor. In this report, we determine the midpoint redox potentials of the Mo-bis-MGD cofactor and FS1–FS4 by EPR spectroscopy, and that of the heme b cofactor by electrochemically induced redox difference spectroscopy. We obtained relatively high values of > 250 mV both for the MoVI–MoV redox couple and the heme b cofactor, whereas FS2 is only reduced at a very low redox potential, causing magnetic coupling with the neighboring FS1 and FS3. We compare the results with the data on related enzymes and interpret their significance for the function of EbDH. Graphical abstract

Comparison of 2-Hydroxyglutarate Detection With sLASER and MEGA-sLASER at 7T

The onco-metabolite 2-hydroxyglutarate (2HG), a biomarker of IDH-mutant gliomas, can be detected with 1H MR spectroscopy (1H-MRS). Recent studies showed measurements of 2HG at 7T with substantial gain in signal to noise ratio (SNR) and spectral resolution, offering higher specificity and sensitivity for 2HG detection. In this study, we assessed the sensitivity of semi-localized by adiabatic selective refocusing (sLASER) and J-difference MEsher-GArwood-semi-LASER (MEGA-sLASER) for 2HG detection at 7T. We performed spectral editing at long TE using a TE-optimized sLASER sequence (110 ms) and J-difference spectroscopy using MEGA-sLASER (TE = 74ms) in phantoms with different 2HG concentrations to assess the sensitivity of 2HG detection. The robustness of the methods against B0 inhomogeneity was investigated. Moreover, the performance of these two techniques was evaluated in four patients with IDH1-mutated glioma. In contrary to MEGA-sLASER, sLASER was able to detect 2HG concentration as low as 0.5 mM. In case of a composite phantom containing 2HG with overlapping metabolites, MEGA-sLASER provided a clean 2HG signal with higher fitting reliability (lower %CRLB). The results demonstrate that sLASER is more robust against field inhomogeneities and experimental or motion-related artifacts which promotes to adopt sLASER in clinical implementations.

Retinal vibrations in bacteriorhodopsin are mechanically harmonic but electronically anharmonic: evidence from overtone and combination bands

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. In spite the light-induced IR difference spectrum between the K intermediate (13-cis retinal) and the initial BR state (all-trans retinal) being first published almost 40 years ago, we present here unreported bands in the 2500 to 1800 cm-1 region. We show that the bands between 2500 and 2300 cm-1 originate from overtone and combination transitions of retinal C-C stretches. We assigned some of the newly reported bands below 2300 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 electronic 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 spectrum by ab initio anharmonic spectral calculations of all-trans retinal in BR, which also reproduced reasonably well the small experimental anharmonic and coupling mechanical constants. Yet, and in spite accounting for both mechanical and electronic anharmonicities, the intensity of overtone C-C transitions was underestimated by a factor of 4 to 20, indicating room for improvement in state-of-the-art anharmonic vibrational calculations.

A microprocessor-aided platform enabling surface differential reflectivity and reflectance anisotropy spectroscopy

Surface differential reflectivity (SDR) and reflectance anisotropy spectroscopy (RAS) [sometimes known as reflectance difference spectroscopy] are two well-known optical spectroscopies used in the investigation of surfaces and interfaces. Their adaptability on different experimental conditions (vacuum, controlled atmosphere and liquid environment) allows for the investigation not only of surface states and/or ultra-thin films but also of more complex interfaces. In these circumstances, the analysis of the sample with both techniques is decisive in view of obtaining a correct picture of the sample optical properties. In this work, we show a microelectronic hardware solution useful to control both a SDR and a RAS apparatus. We describe an electronic architecture that can be easily replicated, and we applied it to a representative sample where the interpretation of the optical properties requires an analysis by both SDR and RAS. Graphic abstract