Oriented immobilization of redox metalloenzymes for direct electron transfer: deciphering loading, orientation, and electronic coupling

Direct electron transfer (DET) in oxidoreductase-based bioelectrocatalysis offers energy efficiency and selectivity over mediated systems. However, realizing its full catalytic potential is limited by an insulating protein matrix surrounding redox cofactors, which necessitates precise electronic coupling and a minimal electron tunneling distance to the electrode surface. This review evaluates strategies for the oriented immobilization of redox metalloenzymes, with the scope limited to studies that meet strict DET criteria, excluding systems in which the observed bioelectrocatalytic signal may originate from a dissociated cofactor acting as a built-in diffusive mediator. Analysis of leading case studies, curated by the chemistry of the enzyme-support interface, reveals that enzyme loading, orientation, and electronic coupling are distinct variables that must be deconvoluted rather than conflated, and that orientation control does not by itself guarantee enhanced performance. These distinctions expose a central bottleneck of the field, namely, the capacity to verify interfacial architecture lags behind the capacity to design it. Empirically, enzyme orientation on surfaces is still assessed predominantly through indirect electrochemical readouts that cannot separate improved alignment from a simple increase in the number of electroactive enzymes. Closing this gap will require operando, single-molecule-resolved characterization benchmarked against dynamic simulation.