Electrochemical\nSmall-Angle X‑ray Scattering\nfor Potential-Dependent Structural Analysis of Redox Enzymes

Various methods exist\nfor exploring different aspects of these\nmechanisms. However, techniques for investigating structural differences\nbetween the reduced and oxidized forms of an enzyme are limited. Here,\nwe propose electrochemical small-angle X-ray scattering (EC-SAXS)\nas a novel method for potential-dependent structural analysis of redox\nenzymes and redox-active proteins. While similar approaches have been\nemployed previously in battery and fuel cell research, biological\nsamples have not yet been analyzed using this technique. Using EC-SAXS,\nwe elucidated the structures of oxidized and reduced bilirubin oxidase\n(BOD). The oxidized BOD favors an open state, enhancing accessibility\nto the active center, whereas the reduced BOD prefers a closed state.\nEC-SAXS not only broadens our understanding of redox enzymes but also\noffers insights that could aid in developing customized enzyme immobilization\nstrategies. These strategies could considerably improve the performance\nof biosensors, biofuel cells, and other bioelectronics.