The cysteine S–H stretching mode reports on long‐range conformational changes in pyruvate oxidase from E. coli

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. The cysteine S–H stretching frequency lies in an exclusive spectral range, and its precise value depends on the H‐bonding environment. Here, we show that Cys‐SH reports directly on structural changes in pyruvate oxidase from E. coli upon redox transition of the flavin cofactor. Though remote from both FAD and the known‐to‐be‐moving C‑terminus, two intrahelical Cys‑SHs sense movements at the dimer interface, possibly indicating structural allostery.