Activation of hydrogen peroxide in horseradish peroxidase occurs within approximately 200 micro s observed by a new freeze-quench device

Abstract

To observe the formation process of compound I in horseradish peroxidase (HRP), we developed a new freeze-quench device with approximately 200 micro s of the mixing-to-freezing time interval and observed the reaction between HRP and hydrogen peroxide (H(2)O(2)). The developed device consists of a submillisecond solution mixer and rotating copper or silver plates cooled at 77 K; it freezes the small droplets of mixed solution on the surface of the rotating plates. The ultraviolet-visible spectra of the sample quenched at approximately 1 ms after the mixing of HRP and H(2)O(2) suggest the formation of compound I. The electron paramagnetic resonance spectra of the same reaction quenched at approximately 200 micro s show a convex peak at g = 2.00, which is identified as compound I due to its microwave power and temperature dependencies. The absence of ferric signals in the electron paramagnetic resonance spectra of the quenched sample indicates that compound I is formed within approximately 200 micro s after mixing HRP and H(2)O(2). We conclude that the activation of H(2)O(2) in HRP at ambient temperature completes within approximately 200 micro s. The developed device can be generally applied to investigate the electronic structures of short-lived intermediates of metalloenzymes.

FIGURE 1

(a) A schematic diagram of the developed freeze-quench device. A solution of 8 mM H₂O₂ in 50 mM MOPS and a solution of 50 mM MOPS were introduced into two 5-ml syringes (S1 and S2, respectively) through sample inlets with membrane filters (I1 and I2). The syringe pump (SP) supplied the respective solutions continuously at a flow rate of 4.2 ml min⁻¹ for each syringe. HRP (400 μM) dissolved in MOPS buffer was introduced in a sample loop (L) through an inlet (I3) and was pushed by the MOPS buffer from syringe S2. Flow valves (V1, V2, and V3) were used to control the sample flow. The H₂O₂ and HRP solutions were introduced into the rapid mixer (R) after they passed though in-line filters (F1 and F2). The mixed solution was flushed against the rotating copper or silver disks (C) immersed in liquid nitrogen dewar (D). Polyethylene plates (K) were placed above the rotating disks to prevent the flow of liquid nitrogen over the rotating disks. (b) A close-up view of the rapid mixer. The two solutions were introduced into the stainless mixing plate (P) and mixed in the T-shaped channel drilled at the end of the plate as depicted in the circle. The plate was sealed with Teflon plates (T1 and T2) and tightened by stainless blocks (M1 and M2).

FIGURE 2

UV-visible absorption spectra of the reaction mixture between horse-heart myoglobin (100 μM) and azide (800 mM) prepared using the freeze-quench device. The spectra were recorded at 120 (trace A), 200 (trace B), 220 (trace C), and 240 K (trace D). The low-temperature absorption spectrum of metmyoglobin powder prepared using the freeze-quench device is also presented for comparison (trace E).

FIGURE 3

UV-visible absorption spectra of the reaction mixture between horse-heart myoglobin in the reduced form (100 μM) and oxygen (∼140 μM) prepared using the freeze-quench device. The spectra were recorded at 130 (trace A), 200 (trace B), 210 (trace C), and 225 K (trace D). The low-temperature absorption spectrum of the ferrous form of myoglobin is also presented for comparison (trace E).

FIGURE 4

UV-visible absorption spectra of the reaction mixture between ferric HRP (20 μM) and H₂O₂ (400 μM) quenched within ∼1 ms after mixing. The sample solution contained 50 mM MOPS adjusted at pH 7.0. The spectra were recorded at 114 (trace A), 160 (trace B), 202 (trace C), 230 (trace D), 246 (trace E), 256 (trace F), and 264 K (trace G). The spectrum of authentic compound I observed at room temperature is also shown (inset).

FIGURE 5

UV-visible absorption spectra of the reaction mixture between ferric HRP (60 μM) and H₂O₂ (10 mM) quenched within ∼1 ms after mixing. The sample solution contained 50 mM sodium phosphate buffer adjusted at pH 7.0. The spectra were recorded at 150 (trace A), 200 (trace B), 220 (trace C), 230 (trace D), 260 (trace E), and 268 K (trace F).

FIGURE 6

The EPR spectra of the reaction mixture between ferric HRP and H₂O₂ quenched within ∼200 μs after the mixing. Trace A: the reference EPR spectrum for the ferric resting state of HRP (400 μM) in 50 mM MOPS buffer at pH 7.0 observed at 5 K. The sample was prepared by using the freeze-quench device. Trace B: the EPR spectrum for the reaction mixture between ferric HRP (400 μM) and H₂O₂ (8 mM) quenched at ∼200 μs in MOPS buffer observed at 5 K. Trace C: the EPR spectrum for the reaction mixture between ferric HRP (400 μM) and H₂O₂ (8 mM) quenched at ∼200 μs in MOPS buffer observed at 15 K. Trace D: the reference EPR spectrum for compound I of HRP prepared by manually freezing the mixture of HRP (310 μM) and H₂O₂ (400 μM) observed at 5 K. The interval between the mixing and freezing was ∼30 s. The buffer solution was 50 mM MOPS at pH 7.0.