# Structural and spectroscopic resolution of the NADPH redox state in the STEAP2 cytosolic oxidoreductase domain

## TL;DR

This paper resolves the redox state of NADPH in the STEAP2 enzyme, providing structural and spectroscopic evidence that clarifies its electron transfer mechanism.

## Contribution

The study provides the first direct structural and spectroscopic validation of NADPH redox state in STEAP2, resolving a key ambiguity in its electron transfer pathway.

## Key findings

- High-resolution crystal structures of STEAP2 OxRD with NADPH were determined, confirming the redox state via single-crystal spectroscopy.
- Conformational differences in the FAD-binding region suggest a model for domain reorientation facilitating FADH2 loading and FAD release.
- The findings clarify a critical ambiguity in STEAP structural biology and highlight the importance of redox-state verification in redox enzyme studies.

## Abstract

The six-transmembrane epithelial antigen of prostate (STEAP) family of membrane proteins comprises four human metalloreductases essential for iron and copper homeostasis, redox balance, and cell proliferation. These enzymes transfer electrons from cytosolic NADPH to extracellular ferric and cupric ions via a FAD and heme-dependent pathway. STEAP2, 3, and four contain an N-terminal cytosolic oxidoreductase domain (OxRD) that enables electron input from NADPH, making STEAP2 ideal for studying redox-state cofactor or cosubstrate dynamics. While recent structures of STEAP proteins have been crucial, the redox state of bound NADPH has remained ambiguous in structural data, limiting mechanistic understanding. Here, we address this key missing piece of ambiguity for understanding the electron transfer pathway. We report high-resolution crystal structures of the STEAP2 OxRD with NADPH, in which the redox state of the cosubstrate is directly validated by single-crystal spectroscopy. Comparison with a recent cryoEM structure reveals conformational differences in the FAD-binding region, suggesting a plausible model in which domain reorientation between the N-terminal OxRD and C-terminal transmembrane domain (TMD) facilitates FADH2 loading and FAD release. These findings resolve a key ambiguity in STEAP structural biology and underscore the importance of experimental redox-state verification in structural studies of redox enzymes.