Cysteine S−H bonds have a spectroscopically convenient stretching frequency of ~2550 cm−1. Their cross-section, however, is low, and the band can be strongly broadened in heterogeneous environments, making detection very challenging. With two-dimensional infrared (2D-IR) setups achieving ever higher sensitivities in recent years, systematic use of the weak Cys−SH absorption band is now within reach, even at low millimolar protein concentrations. Here we demonstrate the capabilities of Cys−SH as an intrinsic 2D-IR label in pyruvate oxidase from E. coli ( EcPOX), an enzyme with ten cysteines in its native sequence. 1D-IR measurements on the wild-type and individual cysteine knock-out variants show that two such residues have especially narrow SH signatures, caused by their intrahelical hydrogen bonding. 2D-IR analysis of these bands reveals an extraordinarily high anharmonicity (~110 cm−1) and a long vibrational lifetime (~4 ps). This allows monitoring spectral diffusion via center line slope analysis for up to 10 ps-separately for both, the ground and excited state. The unique spectroscopic features and its ease of introduction make Cys−SH a useful IR spectroscopic label.

The pyruvate oxidases from Escherichia coli (EcPOX) and Lactobacillus plantarum (LpPOX) are both thiamin‐dependent flavoenzymes. Their sequence and structure are closely related, and they catalyse similar reactions—but they differ in their activity pattern: LpPOX is always highly active, EcPOX only when activated by lipids or limited proteolysis, both involving the protein's C‐terminal 23 residues (the ‘α‐peptide’). Here, we relate the redox‐induced infrared (IR) difference spectrum of EcPOX to its unusual activation mechanism. The IR difference spectrum of EcPOX is marked by contributions from the protein backbone, reflecting major conformational changes. A rare sulfhydryl (−SH) difference signal indicates changes in the vicinity of cysteines. We could pin the Cys–SH difference signal to Cys88 and Cys494, both being remote from the moving α‐peptide and the redox‐active flavin cofactor. Yet, when the α‐peptide is proteolytically removed, the Cys–SH difference signal disappears, together with several difference signals in the amide range. The remaining IR signature of the permanently activated EcPOXΔ23 is strikingly similar to the simpler signature of LpPOX. The loss of the α‐peptide ‘transforms’ the catalytically complex EcPOX into the catalytically ‘simpler’ LpPOX.