Electrochemical Small-Angle X-ray Scattering for Potential-Dependent Structural Analysis of Redox Enzymes

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