Reviewer #1 (Public Review): Mechanism of stepwise electron transfer in six-transmembrane epithelial antigen of the prostate (STEAP) 1 and 2

Abstract

Six transmembrane epithelial antigen of the prostate (STEAP) is a family of four members, and all have a transmembrane domain with a conserved heme binding site. STEAP2 – 4, but not STEAP1, have an intracellular domain that binds to NADPH and FAD. STEAP2 – 4 show metal ion reductase activities, which led to the notion that STEAPs mediate electron transfer starting from NADPH on the intracellular side, through FAD and heme, and finally to a metal ion substrate on the extracellular side. However, the electron transfer chain has not been reconstructed with purified proteins for rigorous investigation. It is also unclear whether STEAP1 has metal ion reductase activity and if so, how electrons are transferred. We expressed and purified mammalian STEAP1 and 2, re-constructed the electron transfer chain in vitro and examined individual electron transfer steps. We find that the heme in both STEAP1 and 2 can transfer electrons to a metal ion substrate and that STEAP1 reduces the metal ion significantly faster than STEAP2. We determined the structure of STEAP2 by cryo-electron microscopy and find that its substrate binding site is less well ordered compared to the structures of STEAP1 and 4, which may explain its lower reductase activity. We also demonstrate that the FAD on STEAP2 becomes diffusible after reduction by NADPH. We show that STEAP1 can form an electron transfer chain with cytochrome b5 reductase. These results establish a solid foundation for understanding the functions of STEAP1 and 2 in cells.