Ca2+ stabilizes the semiquinone radical of pyrroloquinoline quinone

Abstract

Spectroelectrochemical studies were performed on the interaction between Ca(2+) 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 Ca(2+) 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 Ca(2+). In contrast, in the absence of Ca(2+) no semiquinone was observed, either for PQQ in the free state (at pH 7.0) or in the enzyme (holo-X). Incorporation of Ca(2+) 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 26.5 mV) of the two-electron redox potential, indicating that the effect of Ca(2+) 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 Ca(2+) 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.

Studies of Organic Protein Cofactors Using Electron Paramagnetic Resonance

“…38 The wildtype as well as the mutant protein were active in a functional assay using an artificial electron acceptor, implying that the disulfide bridge does not play a role in the general mechanism of the enzyme. The X-band continuous-wave EPR spectrum of the PQQ semiquinone radical in the wild-type QEDH enzyme was found to be slightly asymmetric with a line centered at g iso ¼ 2:0043, and a peak-to-peak line width of 0.5 mT, which is similar to that observed in GDH, 39 in quinohaemoprotein ethanol dehydrogenase, 40 methylamine dehydrogenase (MADH), 41 and in MDH. 42 A slightly larger peak-to-peak line width of 0.7 mT has been reported for MDH.…”

“…The protonation states of the three carboxylic acid groups of PQQ, when bound in quinoproteins, are not known, although in solution at pH 7 it is expected that they would be deprotonated. 39,50 Nevertheless, even if they were protonated, such protons always have negligible hyperfine couplings. The only other positions in the cofactor that could have exchangeable protons with significant hyperfine couplings are at O(4) and O(5).…”

In vitro studies indicate a quinone is involved in bacterial Mn(II) oxidation

“…The role of Ca 2+ may also indicate a more specific role. PQQ requiring enzymes such as methanol dehydrogenase and soluble glucose dehydrogenase (Oubrie et al 1999) require Ca 2+ for binding of PQQ and stabilization of the PQQ semiquinone form (Sato et al 2001). The effect of Ca 2+ on Mn(II) oxidation is consistent with catalysis by a PQQ requiring enzyme.…”

The Electrochemical Reaction Mechanism and Applications of Quinones

“…As a proof, the stable semiquinone radical was observed with EPR and spectroelectrochemistry. A study that shows semiquinone stabilization of pyrroloquinoline quinone (PQQ) cofactor in soluble glucose dehydrogenase by Ca 2+ cation implies the biological significance of this 44 . It is worth making a brief note about the semiquinone radical Q sem , of which stability and properties are of considerable interest because semiquinone radical contributes to reactive oxygen species generation.…”