Ca2+ stabilizes the semiquinone radical of pyrroloquinoline quinone

Spectroelectrochemical studies were performed on the interaction between Ca 2+ and pyrroloquinoline quinone (PQQ) in soluble glucose dehydrogenase (sGD H) and in the free state by applying a mediated continuous-flow column elec trolytic spectroelectrochemical technique. The enzyme forms used were holo- sGDH (the holo-form of sGDH from Acinetobacter calcoaceticus) and an incomp letely reconstituted form of this, holo-X, in which the PQQ-activating Ca2 is lacking. The spectroelectrochemical and ESR data clearly demonstrated t he generation of the semiquinone radical of PQQ in holo-sGDH and in the fre e state in the presence of Ca2+. In contrast, in the absence of Ca2+ no sem iquinone was observed, either for PQQ in the free state (at pH 7.0) or in t he enzyme (holo-X). Incorporation of Ca2+ into the active site of holo-X, y ielding holo-sGDH, caused not only stabilization of the semiquinone form of PQQ but also a negative shift (of 26.5 mV) of the two-electron redox poten tial, indicating that the effect of Ca2+ is stronger on the oxidized than o n the reduced PQQ. Combining these data with the observations on the kineti c and chemical mechanisms, it was concluded that the strong stimulating eff ect of Ca2+ on the activity of sGDH can be attributed to facilitation of ce rtain kinetic steps, and not to improvement of the thermodynamics of substr ate oxidation. The consequences of this conclusion are discussed for the ox idative as well as for the reductive part of the reaction of sGDH.