DOI: 10.5061/dryad.00000008r
title: Kinetics of the redox reactions in STEAP1 and STEAP2

description: The data repository includes the kinetic data of the redox reactions in
 STEAP1 and STEAP2, which are presented in "Mechanism of stepwise
 electron transfer in six-transmembrane epithelial antigen of the prostate
 (STEAP) 1 and 2" by Kehan Chen, Lie Wang, Jiemin Shen, Ah-lim Tsai,
 Ming Zhou and Gang Wu. The data include: 1) the reduction of ferric STEAP1
 by reduced FADH2 and ferrous STEAP2; 2) the reduction of ferric STEAP1 by
 cytochrome b5 reductase; 3) the reduction of ferric STEAP2 with NADPH; and
 4) the oxidation of ferrous STEAP1 and STEAP2 by ferric.NTA.
description: All the data was collected under anaerobic conditions. The
 time-dependent optical changes in the absorbance of the heme in STEAP1 and
 STEAP2 were monitored using either a diode-array UV-Vis spectrophotometer
 or a stopped-flow machine with a rapid-scan accessory. In each spectrum,
 the background absorbance is subtracted. The absorbance at 800 nm is taken
 as the background in the spectra acquired on the spectrophotometer and the
 absorbance at 703–708 nm is taken as the background in the data obtained
 using the stopped-flow rapid-scan method. The time courses of the Soret
 wavelength of the heme were either extracted from the data of the
 time-dependent spectral change or acquired on the stopped-flow machine
 operated in the single wavelength mode.  For more
 experimental details, please refer to the Method section in
 "Mechanism of stepwise electron transfer in six-transmembrane
 epithelial antigen of the prostate (STEAP) 1 and 2" by Kehan Chen,
 Lie Wang, Jiemin Shen, Ah-lim Tsai, Ming Zhou and Gang Wu.
description: # Kinetics of the redox reactions in STEAP1 and STEAP2
 [https://doi.org/10.5061/dryad.00000008r](https://doi.org/10.5061/dryad.00000008r) The kinetics data are stored in Excel files and the filenames correspond to the figures in "Mechanism of stepwise electron transfer in six-transmembrane epithelial antigen of the prostate (STEAP) 1 and 2" by Kehan Chen, Lie Wang, Jiemin Shen, Ah-lim Tsai, Ming Zhou and Gang Wu, https://doi.org/10.7554/eLife.88299.1. In each data file, the title of each sheet indicates the panel in the figure and the reaction. The structures of different types of kinetic data are described as follows. 1. **Time-dependent spectral change** (Figure2, Figure2-FigureSupplement1, Figure3, Figure3-FigureSupplement1, Figure4-FigureSupplement1) The first column is the wavelength and the spectra are listed continuously from column 2. The spectrum acquired at the first time point is always in column 2 and marked "spectrum#1" in the top, and the temporal sequence of the spectra are marked in the top of each column. The acquisition time points of the spectra are listed in the two columns following the last spectrum. 2. **Time courses of the Soret absorbance of ferrous heme** (Figure3-FigureSupplement1, Figure5) The data is presented in two columns, the left one is the time points (in second) and the right is the absorbance at 427 nm (A<sub>427</sub>). 3. **Rate-dependence on the concentration of reduced FADH<sub>2</sub> or ferric.NTA** (Figure2, Figure2-FigureSupplement1, Figure5) The left column is the concentration of reduced FAD (FADH<sub>2</sub>) (in uM) or ferric.NTA (Fe<sup>3+</sup>.NTA) (in uM), followed by the columns of the rate constants, *k*<sub>obs</sub> (in s<sup>-1</sup>) of the fast phase, *k*<sub>obs</sub>\_1 and the slow phase, *k*<sub>obs</sub>\_2. 4. **Bio-layer Interferometry** (Figure3-FigureSupplement2). The profiles of association and dissociation are labeled with the concentration cytochrome *b*<sub>5</sub> reductase (b<sub>5</sub>R) in the top and the response (Red) is listed versus the concentration of *b*<sub>5</sub>R (in uM). The data sheets in each file are listed below. 1. **Figure 2** a. "PanelA STEAP1+FADH2": the spectral change during the reaction of STEAP1 with reduced FAD (FADH<sub>2</sub>); Panel A in Figure 2. b. "PanelBkobs(STEAP1+FADH2)vsFADH2": the dependence of the rate constants, *k*<sub>obs</sub> in the reaction of STEAP1 with FADH<sub>2</sub>, on the concentration of FADH<sub>2</sub>; Panel B in Figure 2. c. "PanelC STEAP1-STEAP2+NADPH": the spectral change in the reaction of the mixture of STEAP1 and STEAP2 with NADPH; Panel C in Figure 2. 2. **Figure 2-FigureSupplement1** "PanelC L230GSTEAP1+FADH2": the spectral change in the reaction of STEAP1 Leu230Gly mutant with reduced FAD (FADH<sub>2</sub>) and the dependence of rate constants, *k*<sub>obs</sub>, on the concentration of FADH<sub>2</sub>; Panel C in Figure 2-figuresupplement1. 3. **Figure 3** a. "PanelA STEAP1-b5R+NADH": the spectral change in the reaction of STEAP1 plus cytochrome b<sub>5</sub> reductase (b<sub>5</sub>R) with NADH; Panel A in Figure 3. b. "PanelB L230GSTEAP1-b5R+NADH": the spectral change in the reaction of STEAP1 Leu230Gly mutant plus b<sub>5</sub>R with NADH; Panel B in Figure 3. 4. **Figure 3-FigureSupplement1** a. "PanelA": the time courses of A<sub>427</sub> in the reaction of STEAP1 only with NADPH and STEAP1 plus b<sub>5</sub>R with NADH; Panel A in Figure 3- figuresupplement1. b. "PanelB" the spectral change in the reaction of STEAP1 plus FAD with NADPH; Panel B-figuresupplement1. 5. **Figure 3-FigureSupplement2** "Sheet1": (1) the bio-layer interferometry profiles in the binding of b<sub>5</sub>R to STEAP1; Panel A in Figure 3-figuresupplement2; (2) the dependence of response on the concentration of b<sub>5</sub>R; Panel B in Figure 3-figuresupplement2. 6. **Figure 4-FigureSupplement1** a. "PanelA STEAP2+NADPH": the spectral change in the reaction of STEAP2 plus FAD with NADPH; Panel A in Figure 4-figuresupplement1. b. "PanelB STEAP2+FADH2": the spectral change in the reaction of STEAP2 with reduced FAD (FADH<sub>2</sub>); Panel B in Figure 4-figuresupplement1. 7. **Figure 5** a. "PanelA&B STEAP1+Fe.NTA": (1) the time courses of A<sub>427</sub> in the reaction of ferrous STEAP1 with Fe<sup>3+</sup>.NTA; Panel A in Figure 5; (2) the dependence of the rate constants, *k*<sub>obs</sub>, on the concentration of Fe<sup>3+</sup>.NTA; Panel B in Figure 5. b. "PanelC&D STEAP2+Fe.NTA": (1) the time courses of A<sub>427</sub> in the reaction of ferrous STEAP2 with Fe<sup>3+</sup>.NTA; Panel C in Figure 5; (2) the dependence of the rate constants, *k*<sub>obs</sub>, on the concentration of Fe<sup>3+</sup>.NTA; Panel D in Figure 5.