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 FADH(2) 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.