Fig. 3: Electrochemical and spectroscopic features of CbA5H^WT and mutagenesis variants.

a Comparison of cyclic voltammograms of CbA5H^WT(Cb-WT), CpI (CpI-WT), and Cb-variant C367D (T = 5 °C, pH 7, 1 atm. of H₂, scan rate 3 mV/s, electrode rotation rate 3000 rpm, currents normalized at E = −0.56 V). b Potential step chronoamperometry of CbA5H^WT and C367D (5 °C, pH 7, 1 atm. of H₂, 1000 rpm). P1: H₂-production current at −0.8 V prior to O₂-exposure; P2: potential step to 0 V; P3: injection of 50 μM O₂; P4: five-fold buffer exchange to re-establish anaerobic conditions; P5: potential step to −0.8 V to measure the residual H₂-production current. c ATR-FTIR-spectroscopy of CbA5H^WT and variant C367D prior and after O₂-exposure (pH 8). d Cyclic voltammograms of CbA5H^WT (black) and mutagenesis variants L364F, P386L, A561F, and L364F-A561F (5 °C, pH 7, 1 atm. of H₂, 20 mV/s, 3000 rpm, currents normalized at E = −0.32 V except WT, at −0.56 V). e Chronoamperometry as in panel B, with CbA5H^WT and the same variants as in d. f ATR-FTIR-spectroscopy (pH 8) of the loop variants, prior, and after O₂-exposure. Electrochemical and IR spectroscopic experiments have been repeated for each protein at 3-4 times with consistent results. Source data are provided in a source data file.