Fig. 5. Whole-cell generation of the H_hyd and H_hydH⁺ states under different conditions probed by EPR spectroscopy. (a) The H_hyd state was observable in EPR spectra recorded on [2Fe]^adt-HydA1 containing cells at pH 7.5, complemented with 10 mM NaDT, flushed with H₂ for 15 min and rapidly frozen (dark yellow spectrum); (b): A new signal attributed to H_hydH⁺ was observed in samples prepared as lane a but at pH 4 (purple spectrum); (c) samples prepared as lane b but excluding NaDT generated a weaker H_hydH⁺ signal (green spectrum); (d) samples prepared as lane b but excluding H₂ flushing did not generate any discernable H_hydH⁺ signal (blue spectrum). The g-values of H_ox/H_oxH (green dashed vertical lines), H_hyd (dark yellow dashed vertical lines) and H_hydH⁺ (purple dashed vertical lines) states are based on the simulations of the spectra (black dash-dotted lines), for details see Fig. S13.† Unassigned weak signals potentially arising from an H_trans-like state indicated with asterisks.⁴⁰ EPR experimental conditions: T = 20 K, P = 1 mW, ν = 9.38 GHz.