Figure 4.

A, SDS-PAGE of purified (lane 1) and deglycosylated (lane 2) CcSDH. B, Effect of PQQ on cytochrome c-reducing activity of CcSDH with L-fucose as a substrate. For the activity measurement in B, 40 nM CcSDH was incubated with 20 mM L-fucose, 50 µM cytochrome c and 1 µM PQQ in HEPES buffer (pH 7.0), and the reduction of cytochrome c was monitored based upon the increase of absorption at 550 nm. C and D, Isothermal titration calorimetry analysis of the PQQ domain of CcSDH and PQQ with the raw data (C) and plots of the integrated peaks (D). The PQQ domain of CcSDH (6.73 µM) was titrated with an initial injection of 2 µL of PQQ (46.3 µM) followed by 69 consecutive injections of 4 µL of PQQ in the presence of 1 mM CaCl₂ and 20 mM sodium acetate (pH 6.0). The black line indicates the best fit to a single-site model.