Ca2+ stabilizes the semiquinone radical of pyrroloquinoline quinone Available to Purchase

Spectroelectrochemical studies were performed on the interaction between Ca²⁺ and pyrroloquinoline quinone (PQQ) in soluble glucose dehydrogenase (sGDH) and in the free state by applying a mediated continuous-flow column electrolytic spectroelectrochemical technique. The enzyme forms used were holo-sGDH (the holo-form of sGDH from Acinetobacter calcoaceticus) and an incompletely reconstituted form of this, holo-X, in which the PQQ-activating Ca²⁺ is lacking. The spectroelectrochemical and ESR data clearly demonstrated the generation of the semiquinone radical of PQQ in holo-sGDH and in the free state in the presence of Ca²⁺. In contrast, in the absence of Ca²⁺ no semiquinone was observed, either for PQQ in the free state (at pH7.0) or in the enzyme (holo-X). Incorporation of Ca²⁺ into the active site of holo-X, yielding holo-sGDH, caused not only stabilization of the semiquinone form of PQQ but also a negative shift (of 26.5mV) of the two-electron redox potential, indicating that the effect of Ca²⁺ is stronger on the oxidized than on the reduced PQQ. Combining these data with the observations on the kinetic and chemical mechanisms, it was concluded that the strong stimulating effect of Ca²⁺ on the activity of sGDH can be attributed to facilitation of certain kinetic steps, and not to improvement of the thermodynamics of substrate oxidation. The consequences of this conclusion are discussed for the oxidative as well as for the reductive part of the reaction of sGDH.