Stopped-flow kinetics measurements.

The reactions of ferrous rSTEAP1 with ferric or cupric ion complexes, Fe³⁺•EDTA, Fe³⁺•citrate and Cu²⁺•EDTA, or O₂ were studied using an Applied Photophysics (Leatherhead, UK) model SX-18MV stopped-flow instrument, following the spectral changes using a diode-array detector and the time courses of single wavelength(s) using a monochromator to minimize photodecomposition. The sample handling unit was kept in an anaerobic chamber by Coy Lab Products, Inc. (Grass Lake, MI). Ferrous rSTEAP1 was prepared by Na₂S₂O₄ titration after five cycles of vacuum (30 s/cycle) and argon displacement (5 min/cycle) in a tonometer. Diode-array data were analyzed with the global analysis method using the Pro-K package (Applied Photophysics). Observed rate, k_obs, of single wavelength data were obtained by fitting the time courses of optical changes to the standard exponential functions:

where A and A₀ are the absorbance and final absorbance at equilibrium, respectively; k_obs1 and k_obs2 are the observed rates of the two phases; a₁ and a₂ are the amplitudes of each phase; t is the reaction time.

The k_obs of some of the rSTEAP1 reactions with Fe³⁺•EDTA or Fe³⁺•citrate showed linear dependence on the concentrations of ferric ion complex, the 2^nd order rate constants were obtained based on the linear dependence of k_obs on [Fe³⁺]:

where the slope represents 2^nd order forward reaction constant; k_f, and the y-intercept approximates the rate constant of the reverse reaction, k_r; [Fe³⁺] is the concentration of [Fe³⁺•EDTA] or [Fe³⁺•citrate].