Electron relay via diffusible and protein-bound flavin cofactor in human six-transmembrane epithelial antigen of the prostate (STEAP)

Six transmembrane epithelial antigen of the prostate (STEAP) is a family of membrane-embedded metal ion reductases that transfer electrons across the cell membranes. STEAPs are unique to mammals and implicated in metabolic and inflammatory responses and are significantly upregulated in many types of cancer cells. There are four members in the family, STEAP1 - 4, and all STEAPs have a transmembrane domain (TMD) that has a conserved heme binding site, and STEAP2 - 4, but not STEAP1, have an intracellular reductase domain (RED) that binds to NADPH and FAD. NADPH, FAD, and heme form an electron transfer chain that allows electron flow across the cell membranes, however, the mechanism of the stepwise cross-membrane electron transfer remains unclear. It is also unclear how STEAP1, which does not have a RED, acquires and transfers electrons. We expressed and purified human STEAP2 (hSTEAP2), and constructed the electron transfer chain in vitro. Purified hSTEAP2 mediates electron transfer from NADPH to FAD and to heme, with a NADPH oxidation rate of 0.0026 per second. The time course for reduction of heme is more complex with an initial rate of ~ 0.00016 per second. We also found that the heme in hSTEAP2 has a low-spin electron structure and thus a rigid coordination, which is consistent with its high occupancy in the purified protein and its role as part of the electron transfer chain. We then determined the structure of hSTEAP2 in complex with NADP, FAD, and heme by cryo-electron microscopy to 3.2 Å. Human STEAP2 forms a homotrimer and its structure is similar to that of hSTEAP4. NADP+, FAD, and heme are well-resolved in the structure, and while the current conformation would allow electron transfer from FAD to heme, the FAD isoalloxazine ring is ~ 19 Å away from NADPH and does not support hydride transfer. Significant structural changes are required to accommodate dissociation of the FAD isoalloxazine ring from the TMD such that the FAD may become diffusible after its reduction. To test this hypothesis and also to find out how STEAP1 may transfer electrons, we reconstructed an electron transfer chain for STEAP1 and found that the heme in STEAP1 can be reduced by FAD produced either by the full-length STEAP2 or by the soluble RED domain from STEAP4. These results support a diffusible FAD mechanism and demonstrate that STEAP1 is capable of mediating electron transfer across the cell membranes. In summary, our study established a structural and functional framework for further analyses for resolving the mechanism of electron transfer in STEAPs.