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

Abstract

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.

Figure 1

EC-SAXS cell and measurement setup. a Photograph of cell components: polyimide films with screen-printed electrodes, a comb-shaped epoxy resin spacer (thickness 1.5 mm), and front and back epoxy resin parts with windows for X-rays and holes for mounting on the sample holder. b Photograph of the EC-SAXS cell setup in the measurement chamber connected to a potentiostat. c Photograph of the assembled EC-SAXS cell. d Schematic showing a cross-section of the EC-SAXS cell during measurement.

Figure 2

SAXS intensity data and distance distribution for oxidized and reduced BOD (50 mg/mL). a Scattering intensity for electrochemically (EC) and chemically (chem) oxidized and reduced BOD, presented in a double-logarithmic plot. Scattering profiles were shifted along the intensity axis to enhance visibility. b Distance distribution function P(r) for electrochemically oxidized BOD at 0.8 V vs Ag/AgCl. c Distance distribution P(r) for electrochemically reduced BOD at −0.1 V vs Ag/AgCl.

Figure 3

Ab initio models of oxidized and reduced BOD. (Damaver) The beads are color-coded to indicate spatial positioning, with red beads in front and green/blue beads in the background; 50 mg/mL BOD samples. a Electrochemically oxidized BOD. D_max = 7.82 nm. b Electrochemically reduced BOD. D_max = 6.80 nm. c Chemically oxidized BOD. D_max = 7.49 nm. d Chemically reduced BOD. D_max = 6.73 nm.

Figure 4

Refined high-resolution models of oxidized and reduced BOD. Models are derived from PDB entry 6IQZ; 50 mg/mL BOD samples. a Electrochemically oxidized BOD with an open structure. T1 Cu, T2/T3 site, Asn-197, and Asn-394 are marked. b Electrochemically reduced BOD with a closed structure. T1 Cu, T2/T3 site, Asn-197, and Asn-394 are marked. c Chemically oxidized BOD with an open structure. d Electrochemically oxidized BOD with the ab initio model as an envelope. e Chemically reduced BOD with a closed structure. f Electrochemically reduced BOD with the ab initio model as an envelope.