Mid-IR spectroscopy is a powerful and label-free technique to investigate protein reactions. In this study, we use quantum-cascade-laser-based dual-comb spectroscopy to probe protein conformational changes and protonation events by a single-shot experiment. By using a well-characterized membrane protein, bacteriorhodopsin, we provide a comparison between dual-comb spectroscopy and our homebuilt tunable quantum cascade laser (QCL)-based scanning spectrometer as tools to monitor irreversible reactions with high time resolution. In conclusion, QCL-based infrared spectroscopy is demonstrated to be feasible for tracing functionally relevant protein structural changes and proton translocations by single-shot experiments. Thus, we envisage a bright future for applications of this technology for monitoring the kinetics of irreversible reactions as in (bio-)chemical transformations.

The recently discovered photo-activated adenylyl cyclase (mPAC from Microcoleus chthonoplastes) is the first PAC that owes a light-, oxygen- and voltage-sensitive (LOV) domain for blue-light sensing. The photoreaction of the mPAC receptor was studied by time-resolved UV/vis and light-induced Fourier transform infrared (FTIR) absorption difference spectroscopy. The photocycle comprises of the typical triplet state LOV and the thio-adduct state LOV . While the adduct state decays with a time constant of 8 s, the lifetime of the triplet state is with 656 ns significantly shorter than in all other reported LOV domains. The light-induced FTIR difference spectrum shows the typical bands of the LOV and LOV intermediates. The negative S-H stretching vibration at 2573 cm is asymmetric suggesting two rotamer configurations of the protonated side chain of C194. A positive band at 3632 cm is observed, which is assigned to an internal water molecule. In contrast to other LOV domains, mPAC exhibits a second positive feature at 3674 cm which is due to the O-H stretch of a second intrinsic water molecule and the side chain of Y476. We conclude that the latter might be involved in the dimerization of the cyclase domain which is crucial for ATP binding.

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