Reduction of the heme on STEAP1.

(A) Rapid-scan reaction of 1.1 μM STEAP1 with 4.5 μM reduced FAD (FADH−); the spectral change was monitored for 20 s. (B) The time course of A427 (black), the Soret absorbance of ferrous heme, was extracted from the rapid-scan data. Red: biphasic exponential fit with rate constants kobs of 7.7 (± 0.30) and 0.67 (± 0.034) s−1, respectively (n = 3). The percentage of each phase is 60% and 40%, respectively. Inset, the dependence of rate constants on [FADH−]. Dot, the fast phase; triangle, the slow phase. Lines, fit with equation kobs = vmax * [FADH−]/(KM + [FADH−]). (C) The spectral changes in the reaction of a mixture of 1.1 μM STEAP2 and 0.9 μM STEAP1 (plus 2.2 μM FAD) with 60 μM NADPH; the spectral change was monitored for 1 hr. The direction of the spectral changes is indicated by the arrows. Blue, the spectrum captured at the start of the reaction; red, the spectrum after 1 hour reaction. Inset, the resolved spectral species by deconvolution and the conversion rate constant. Black, ferric STEAP and red, ferrous STEAP.

Reduction of STEAP1 by cytochrome b5 reductase.

(A) The rapid-scan reaction of 1.5 μM STEAP1 and 1.5 μM cytochrome b5 reductase (b5R) with 10 μM NADH; the spectral change was monitored for 20 s. The arrows indicate the direction of the spectral change. Inset: the resolved spectral species and the conversion rate constants. Black, ferric STEAP1with b5R, red, a spectral intermediate, and green, ferrous STEAP1with fully reduced b5R. (B) L230G STEAP1 and b5R were reacted with 10 μM NADH; the spectral change was monitored for 50 s. The direction of spectral change is indicated by the arrows. Inset, the resolved spectral species by deconvolution and the rate constants. Inset: black, ferric L230G STEAP1 with b5R, red, a spectral intermediate, and green, ferrous L230G STEAP1 with fully reduced b5R.

Reduction of Fe3+-NTA by ferrous STEAP1 and STEAP2.

(A) The time courses of A427 in the reactions of 1.1 μM ferrous STEAP1 with 25 (black), 75 (red), 125 (green), 175 (yellow), and 175 μM Fe3+-NTA (blue). The rate constants, kobs, are estimated by biphasic exponential fit to the time courses. One of such fits is shown by the black line. (B) Dependence of the rate constants kobs on [Fe3+-NTA]. Circles, kobs of the fast phase of the A427 time courses; triangles, kobs of the slow phase. (C) The time courses of A427 in the reactions of 1.1 μM ferrous STEAP2 with 75 (black), 125 (red), and 175 μM Fe3+-NTA (green). The time courses in the initial 2 s of the reactions are fitted with a biphasic exponential function, and one such fit is shown by the black line. (D) The rate constants estimated for the initial 2 s, kobs, are plotted versus [Fe3+-NTA]. Circles, kobs of the fast phase of the A427 time courses; triangles, kobs of the slow phase. At reaction time longer than 2 s, the time courses in (C) show more complicated kinetics and no clear dependence on [Fe3+-NTA].

Cryo-EM structure of STEAP2.

The sharpened density map (A) and cartoon presentation (B) for STEAP2 homotrimer. Top, the side view of STEAP2 homotrimer, and the grey bar represents the membrane; “in”, the intracellular side and “out’, the extracellular side. Bottom, the top view of STEAP2 homotrimer from the extracellular side. (C) The structure of one STEAP2 protomer (cartoon) with the prosthetic group heme and the cofactors FAD and NADP+ (sticks). Left, side view and right, top view from the extracellular side. (D) The topographic representation of the secondary structural elements. The α helices and β strands are represented by bars and arrows respectively. Dashed lines represent the unresolved segments. (E) The schematic representation of the spatial relationship of NADP+, FAD, and heme, shown as sticks. Trp152 and Leu371 are also shown as sticks. TMD is represented as the outline with grey shade and the OxRD with pink shade.

Electron transfer in STEAP1 and STEAP2.

NADPH (blue) and FAD (orange) bind to the OxRD in STEAP2 (A, olive shade) with the nicotinamide ring of NADPH aligned with the isoalloxazine ring of FAD for hydride transfer (B). The reduced FAD adopts the extended conformation with its isoalloxazine ring bound deep in the TMD of STEAP2 (C, teal shade) or dissociates from the OxRD to bind STEAP1 (D ➔ E, cyan shade) and transfers electrons to heme (salmon). NADP(H) and FAD(H2) are co-factors that associate with and dissociate from the STEAP protein in each redox cycle while the heme, as a prosthetic group, stays bound to the protein. Cytochrome b5 reductase (F, sand shade) docks on STEAP1 from the intracellular side, forming a complex for electron transfer (G). The FAD-to-heme electron transfer in STEAP is likely mediated through a bulky sidechain (purple), Leu230 in STEAP1 and Leu371 in STEAP2, respectively.