Investigating the Impact of Multi-Heme Pyrroloquinoline Quinone-Aldehyde Dehydrogenase Orientation on Direct Bioelectrocatalysis via Site Specific Enzyme Immobilization

To investigate the effect of the orientation of large and complex multi-subunit enzymes (MW > 50 kDa) on direct bioelectrocatalysis, we immobilized enzymes known to be capable of direct electron transfer (DET) via a site specific immobilization technique to form a monolayer of biocatalysts with a uniform orientation toward the gold electrode. Six recombinant pyrroloquinoline quinone-dependent aldehyde dehydrogenases (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. His-tags were utilized as linking sites to perform site specific immobilization of PQQ-AlDHs. Results show that the orientation of multi-subunit enzymes can affect DET greatly by varying the electron tunneling distances. The favorable orientation allowing for a minimal heme c electron transfer distance showed a DET rate 6.6-fold higher than that with the orientation closest to the active site of the enzyme, while the unfavorable attachment to a nonelectroactive subunit showed no DET.