Investigating the Role of Orientation of PQQ-Dependent Dehydrogenases on Electrochemical Performance of a Bioanode

PQQ-dependent multi-heme containing aldehyde dehydrogenase (PQQ-AlDH) has been previously demonstrated to be capable of performing direct electron transfer (DET). However, theory predicts that the rate of direct bioelectrocatalysis for complex multi-subunit proteins such as PQQ-AlDH is closely related to the proximity and orientation of the enzymes toward the electrode surface, which determines the electron tunneling distances and the current density. In order to investigate the impact of enzyme orientation on the catalysis rate, we immobilized PQQ-AlDH via a site specific immobilization technique to form a monolayer of biocatalysts with a uniform orientation on a gold electrode. Six recombinant PQQ-AlDHs were employed, where the enzymes had been labeled with six-histidine tags (His-tag) at the N- or C-terminus of each of the three subunits. These His-tags were utilized as linking sites to perform site specific isotropic immobilization of PQQ-AlDHs. Results show that the orientation of PQQ-AlDHs can affect direct biocatalysis rate greatly by varying the electron tunneling distances. The favorable orientation with a minimal heme c electron transfer distance showed a current density that is 6.6-fold higher than the electrode with the orientation closest to the active site of the enzyme, while the unfavorable attachment to a non-electroactive subunit showed no bioelectrocatalytic current.