Redox Properties of Pyrroloquinoline Quinone in Pyranose Dehydrogenase Measured by Direct Electron Transfer

Redox proteins regulate the chemical properties of cofactors through the protein environment of the active site. The redox potentials and pK<inf>a</inf> values of the cofactors within the protein are crucial factors in determining the mechanism as they control the electron and proton transfer rates, respectively. The laborious determination of potentials and pK<inf>a</inf> values using spectroscopic techniques can be avoided using direct electrochemistry. The aim of this work was to determine the redox potentials and pK<inf>a</inf> values of pyrroloquinoline quinone (PQQ) in an enzyme with direct electron transfer (DET) using protein film voltammetry. While the redox properties of PQQ in aqueous and organic media have been studied, limited results of PQQ in an enzyme active site are available. Analysis of PQQ-dependent enzymes with direct electron transfer (DET) has been particularly difficult to establish. Here, we successfully observed redox waves of PQQ bound to the PQQ domain from the fungal PQQ-dependent pyranose dehydrogenase with a gold nanoparticle-modified electrode and determined the redox potentials at various pH values. Electron paramagnetic resonance (EPR) spectroscopy showed the formation of a PQQ semiquinone radical at an applied potential between the potentials of PQQ<inf>ox</inf>/PQQ<inf>sem</inf> and PQQ<inf>sem</inf>/PQQ<inf>red</inf>. These results clearly show that two-step one-electron transfer takes place via a semiquinone intermediate in the DET reaction. Both potentials shifted depending on pH; however, deviations from the 1e^-/1H⁺ behavior were observed at high pH. The pK<inf>a</inf> of PQQH^-/PQQH<inf>2</inf> was determined to be close to neutral compared with a much higher value for free PQQ. At the physiological pH of this enzyme, which is neutral to slightly alkaline, the fully reduced form of PQQ was deprotonated, and both one-electron processes are very close in potential. This ensures a smooth energy landscape for optimal catalysis. Overall, these results show that the protein environment carefully tunes the properties of the PQQ cofactor for efficient catalysis involving both two-electron and one-electron chemistry.