Re-oxidation of ferrous STEAP2 by Fe3+-NTA.

(A) The time courses of A427 in the reactions of 1.1 μM STEAP2(Fe(II)) with 75 (black), 125 (red), and 175 μM Fe3+-NTA (green). (B) Comparison between the kinetics of STEAP2(Fe(II)) and STEAP1(Fe(II)) with 125 μM Fe3+-NTA.

Heme reduction in STEAP2.

(A) STEAP2, 2.3 μM, is pre-incubated with 2.5 μM FAD and reacted anaerobically with 45 μM NADPH. The direction of spectral change is indicated by the arrows. Inset, the resolved spectral species by deconvolution and the conversion rate constant. Black: STEAP2(Fe(III)) with FAD and red, STEAP2(Fe(II)) with reduced FAD.

Anaerobic rapid-scan reaction of STEAP1 with reduced FAD.

(A) Rapid-scan reaction of 1.1 μM STEAP1 with 4.5 μM pre-reduced FAD. (B) The time course of A427, the Soret absorbance of ferrous heme, extracted from rapid-scan data. Red: biphasic exponential fit with rate constants of 7.7 (± 0.30) and 0.67 (± 3.4) × 10-2 s-1, respectively (n = 3). Inset, the dependence of the fast phase (dot) and slow phase (triangle) rate constants on [FAD]. Lines, fit with equation k = kmax * [FAD]/(KM + [FAD]).\

Reduction STEAP1 by FAD-dependent reductases.

(A) The rapid-scan reaction of 1.5 μM STEAP1 and 1.5 μM b5R with 10 μM NADH. The arrows indicate the direction of the spectral change. Inset: the resolved spectral species and the conversion rate constants. Black, STEAP1(Fe(III))/b5R; red, a spectral intermediate, and green, STEAP1(Fe(II))/reduced b5R. (B) 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 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 hr reaction. Inset, the resolved spectral species by deconvolution and the conversion rate constant. Black, STEAP(Fe(III)) and red, STEAP(Fe(II)).

Electron transfer pathways in STEAP1 and 2.

(A) The FAD binds STEAP2 in an extended conformation straddling the TMD (grey) and RED (pink) domains of different protomers. The heme binds in the TMD and NADP+ binds in the RED. (B) The hypothetic “folded” conformation of FAD stacks the isoalloxazine ring with the NADPH nicotinamide ring for hydride transfer. The switch between the two FAD conformations (purple arrow) is the “FAD-shuttling” mechanism (red arrow).13 Significant structural changes may be required to accommodate the conformational switch of the FAD and such structural changes may allow the dissociation and rebinding of reduced FAD. (C) The reduced FAD generated in the REDs of STEAP2 or b5R can reduce STEAP1 (blue arrow). Heme, yellow; FAD, orange; NAD(P)H, green.

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. Left, side view and right, top view from the extracellular side. (d) The topographic representation of the secondary structural elements. The 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. TMD, grey shade and RED, pink shade.

The FAD in STEAP2.

The FAD (orange) binds STEAP2 in an extended conformation. Its isoalloxazine protrudes deep into the TMD domain (green) and the adenine ring stacks with Trp152 (pink) from the RED of a neighboring protomer (olive). Leu371 (purple) lies halfway between the isoalloxazine ring and heme (red, iron atom in yellow). NADP+ in the RED is in blue.

Reduction of L230G STEAP1 by b5R.

(A) L230G STEAP1 and b5R were reacted with 10 μM NADH. The direction of spectral change is indicated by the arrows. Inset, the resolved spectral species by deconvolution and the rate constants. Inset: black, L230G STEAP1(Fe(III)/b5R, red, a spectral intermediate, and L230G STEAP1(Fe(II))/reduced b5R.