Mechanism of stepwise electron transfer in six-transmembrane epithelial antigen of the prostate (STEAP) 1 and 2

Six transmembrane epithelial antigen of the prostate (STEAP) is a family of membrane-embedded hemoproteins with four members, STEAP1-4, all of which have a transmembrane domain (TMD) that chelates a heme prosthetic group. STEAP2-4, but not STEAP1, have an intracellular oxidoreductase domain (OxRD) so that an electron transfer chain composed of NADPH, FAD, and heme is established to mediate electron transfer across cell membranes. However, it is not known whether STEAP1 can establish a physiologically relevant electron transfer chain. Here we show that reduced FAD binds to STEAP1 and enables reduction of the heme. We also show that a soluble cytochromeb₅reductase can dock on STEAP1 and serve as a surrogate OxRD to reduce the heme. These results provide the first evidence that STEAP1 can support a cross-membrane electron transfer chain. It is not clear whether FAD, which relays electrons from NADPH to heme and interacts with both OxRD and TMD, remains constantly bound to the STEAPs. We found that FAD reduced by STEAP2 can be utilized by STEAP1, supporting the hypothesis that FAD is diffusible rather than staying bound to STEAP2. We determined the structure of human STEAP2 in complex with NADP⁺and FAD to an overall resolution of 3.2 Å by cryo-electron microscopy. The structure of STEAP2 shows that the two cofactors bind similarly to those in the STEAP4 structure and thus a diffusible FAD is likely a general feature of the electron transfer mechanism in the STEAPs. The structure of STEAP2 also shows that its extracellular regions are less structured than those of STEAP4 or STEAP1, and further experiments show that STEAP2 reduces Fe³⁺-NTA with a rate significantly slower than STEAP1. These results establish a solid foundation for understanding the function and mechanisms of STEAP family of proteins.