Characterization of the peroxidase mechanism upon reaction of prostacyclin synthase with peracetic acid. Identification of a tyrosyl radical intermediate

Abstract

Prostacyclin synthase (PGIS) is a membrane-bound class III cytochrome P450 that catalyzes an isomerization of prostaglandin H(2), an endoperoxide, to prostacyclin. We report here the characterization of the PGIS intermediates in reactions with other peroxides, peracetic acid (PA), and iodosylbenzene. Rapid-scan stopped-flow experiments revealed an intermediate with an absorption spectrum similar to that of compound ES (Cpd ES), which is an oxo-ferryl (Fe(IV)O) plus a protein-derived radical. Cpd ES, formed upon reaction with PA, has an X-band (9 GHz) EPR signal of g = 2.0047 and a half-saturation power, P(1/2), of 0.73 mW. High-field (130 GHz) EPR reveals the presence of two species of tyrosyl radicals in Cpd ES with their g-tensor components (g(x), g(y), g(z)) of 2.00970, 2.00433, 2.00211 and 2.00700, 2.00433, 2.00211 at a 1:2 ratio, indicating that one is involved in hydrogen bonding and the other is not. The line width of the g = 2 signal becomes narrower, while its P(1/2) value becomes smaller as the reaction proceeds, indicating migration of the unpaired electron to an alternative site. The rate of electron migration ( approximately 0.2 s(-1)) is similar to that of heme bleaching, suggesting the migration is associated with the enzymatic inactivation. Moreover, a g = 6 signal that is presumably a high-spin ferric species emerges after the appearance of the amino acid radical and subsequently decays at a rate comparable to that of enzymatic inactivation. This loss of the g = 6 species thus likely indicates another pathway leading to enzymatic inactivation. The inactivation, however, was prevented by the exogenous reductant guaiacol. The studies of PGIS with PA described herein provide a mechanistic model of a peroxidase reaction catalyzed by the class III cytochromes P450.

Figure 1

Stopped-flow experiments of the reaction of PGIS (110 μM) with PA (1.1 mM) at 23 °C using a 2 mm light path. (A) Rapid-scan spectra were recorded at 0.0013, 0.129, 0.257, 0.385, 0.513, and 1.0 s after mixing. Arrows indicate the direction of spectral changes with increasing time. Inset: Eexpansion of data in the visible region. (B) Spectral intermediates were resolved by a two-step model A → B → C using ProK analysis. The spectrum for species A is shown with a solid line, B with a dashed line, and C with a dotted line in the Soret region and the visible region (inset). (C) Secondary plots of the observed rates determined at 418 nm versus PA concentration were analyzed for the first phase (filled circles) and the second phase (open circles). The inset shows the kinetic traces for reactions of PGIS with 35 (top), 70, 140, 280, and 420 (bottom) μM PA.

Figure 2

Stopped-flow experiments of the reaction of PGIS (10 μM) with PhIO (250 μM) at 23 °C using a 1 cm light path. (A) Rapid-scan spectra were recorded at 0.0013, 0.104, 0.206, 0.308, 0.411, 0.513, 0.616, 0.718, 0.821, 0.923, and 1.02 s. Arrows indicate the direction of spectral changes with increasing time. (B) Spectral intermediates A′ (solid line), B′ (dotted line), and C′ (dashed line) were resolved by a two-step model A′ → B′ → C′ over 0.5 s reaction using global analysis. (C) Left panel: Time courses of PGIS reacted with 51 (top), 80, 115, 166, 224, 320, and 480 (bottom) μM PhIO. The kinetic traces of the initial 0.2 s are shown in the inset. Right panel: Secondary plots of the observed rates determined at 418 nm versus PhIO concentration are shown for the first phase (filled circles), the second phase (open circles), and the third phase (filled triangles).

Figure 3

X-band EPR spectra of the reaction of PGIS with PA. Resting PGIS (150 μM manually mixed with 4 volumes of NaP_i buffer) or samples collected after PGIS (150 μM) was mixed with PA (1.5 mM) for 8 ms, 100 ms, 1 s, and 10 s. EPR conditions were microwave power, 4 mW; microwave frequency, 9.6 GHz; modulation amplitude, 10.87 G; and temperature, 10 K. Numbers above the vertical arrows are the g values of the rhombic low-spin heme center.

Figure 4

Progressive EPR power saturation for the radical intermediate trapped at 100 ms reaction of PGIS (150 μM) with PA (1.5 mM). EPR parameters were microwave power, 12.9 μW–25.8 mW; microwave frequency, 9.3 GHz; modulation amplitude, 2 G; and temperature, 110 K. Arrow indicates the direction of increasing microwave power. Lower left inset: microwave saturation curve of the g = 2 radical. The P_1/2 value, fitted by eq 2 in which b was given as 1, is 0.73 mW. Upper right inset: Experimental (solid line) and simulated (dashed line; using the parameters in Table 2) X-band spectra of the freeze-quenched sample collected by reaction of 700 μM PGIS with 7 mM PA for 25 ms. Microwave power, 0.1 mW; microwave frequency, 9.3 GHz; modulation amplitude, 1 G; and temperature, 110 K.

Figure 5

D-band (130 GHz) EPR spectra of 240 μM PGIS reacted with a 10-fold excess PA for 100 ms and freeze quenched in <1 s. Exp, the experimental spectrum (solid line); Sim, the simulated spectrum (dash line) using the parameter values shown in Table 2. The experimental spectrum is the first derivative of an average of five echo-detected spectra obtained at 15 K with ~22 min total acquisition time and parameters described in Materials and Methods.

Figure 6

Kinetics of g = 2 radical (closed circle), residual low-spin heme (opened circle), and g = 6 high-spin heme (closed triangle) EPR signals in the reaction of PGIS (150 μM) with PA (1.5 mM). Data were analyzed as described in the Results.

Figure 7

Plot of line width versus reaction time. Data were fitted using one-exponential decay. Inset: Narrow scan EPR spectra of g = 2 radical of the reaction between PGIS (150 μM) and PA (1.5 mM) collected at 8 ms, 100 ms, 1 s, and 10 s. EPR parameters were microwave power, 1 mW; microwave frequency, 9.3 GHz; modulation amplitude, 2 G; and temperature, 110 K.

Figure 8

The peroxidase reaction mechanism for PGIS with PA and PhIO. The boxes in broken line represent the putative intermediates not observed in this study.

Figure 9

(A) The position of tyrosyl radical candidates nearby the PGIS heme (PDB code 2IAG). Distances of the two most likely candidates, Tyr259 and Tyr446, from the heme are indicated. (B) Molecular structure of the tyrosyl radical with atomic numbering (left) and end-on view along the C1–C_β bond showing the dihedral angle, θ, and the orientation of the β protons.