Oxyferryl heme and not tyrosyl radical is the likely culprit in prostaglandin H synthase-1 peroxidase inactivation

Abstract

Prostaglandin H synthase-1 (PGHS-1) is a bifunctional heme protein catalyzing both a peroxidase reaction, in which peroxides are converted to alcohols, and a cyclooxygenase reaction, in which arachidonic acid is converted into prostaglandin G2. Reaction of PGHS-1 with peroxide forms Intermediate I, which has an oxyferryl heme and a porphyrin radical. An intramolecular electron transfer from Tyr385 to Intermediate I forms Intermediate II, which contains two oxidants: an oxyferryl heme and the Tyr385 radical required for cyclooxygenase catalysis. Self-inactivation of the peroxidase begins with Intermediate II, but it has been unclear which of the two oxidants is involved. The kinetics of tyrosyl radical, oxyferryl heme, and peroxidase inactivation were examined in reactions of PGHS-1 reconstituted with heme or mangano protoporphyrin IX with a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), and ethyl hydrogen peroxide (EtOOH). Tyrosyl radical formation was significantly faster with 15-HPETE than with EtOOH and roughly paralleled oxyferryl heme formation at low peroxide levels. However, the oxyferryl heme intensity decayed much more rapidly than the tyrosyl radical intensity at high peroxide levels. The rates of reactions for PGHS-1 reconstituted with MnPPIX were approximately an order of magnitude slower, and the initial species formed displayed a wide singlet (WS) radical, rather than the wide doublet radical observed with PGHS-1 reconstituted with heme. Inactivation of the peroxidase activity during the reaction of PGHS-1 with EtOOH or 15-HPETE correlated with oxyferryl heme decay, but not with changes in tyrosyl radical intensity or EPR line shape, indicating that the oxyferryl heme, and not the tyrosyl radical, is responsible for the self-destructive peroxidase side reactions. Computer modeling to a minimal mechanism was consistent with oxyferryl heme being the source of peroxidase inactivation.

FIGURE 1

Peroxidase mechanism of FePGHS-1 and MnPGHS-1 with hypothetical self-inactivation pathways, adapted from Dietz et al. (4) and Wu et al. (9). Fe⁴⁺=O/P^+•, oxyferryl heme with a porphyrin cation radical, or Mn⁵⁺ (Intermediate I); Fe⁴⁺=O/P or Mn⁴⁺=O/P, Compound II; Fe⁴⁺=O/P or Mn⁴⁺=O/P + Tyr^• (in dashed box), Intermediate II; ROOH, peroxide; ROH, alcohol; Red, reducing equivalent from cosubstrate; Red_o, reacted cosubstrate. Compound II and Tyr^• are shown as discrete oxidized components of Intermediate II to emphasize their redox independence. The oxyferryl heme of Compound II is proposed to lead to inactive forms of enzyme (E_III and E_in).

FIGURE 2

EPR spectra of the tyrosyl radical formed during reaction of Fe-PGHS-1 with 15-HPETE. Fe-PGHS-1 samples (15 µM) in 100 mM KP_i (pH 7.2), 0.04% octyl glucoside, and 10% glycerol was reacted at room temperature with 5 equiv of 15-HPETE for the indicated times before freeze trapping. The arrow indicates the direction of intensity changes with time. EPR conditions: microwave power, 4 mW; frequency, 9.23 GHz; modulation amplitude, 3.2 G; temperature, 98 K.

FIGURE 3

Kinetics of tyrosyl radical and oxyferryl heme levels during reaction of Fe-PGHS-1 with 15-HPETE. Fe-PGHS-1 (15 µM) in 100 mM KP_i (pH 7.2), 0.04% octyl glucoside, and 10% glycerol was reacted at room temperature with either 5 (A) or 2 equiv (B) of 15-HPETE. Tyrosyl radical levels were determined by double integration of EPR signals such as those shown in Figure 2 (●), and oxyferryl heme levels were followed by A₄₂₈ in parallel stopped-flow experiments (─). Dashed lines indicate exponential fits for tyrosyl radical formation and decay phases.

FIGURE 4

EPR spectra of the tyrosyl radical formed during reaction of Mn-PGHS-1 with 15-HPETE. Mn-PGHS-1 samples (40 µM) in 100 mM KP_i (pH 7.2), 0.04% octyl glucoside, and 10% glycerol were reacted at room temperature with 2 equiv of 15-HPETE for the indicated times before freeze trapping. The arrow indicates the direction of intensity changes with time. EPR conditions are the same as those described in the legend of Figure 2.

FIGURE 5

Kinetics of tyrosyl radical and Mn⁵⁺=O levels during reaction of Mn-PGHS-1 with 15-HPETE. Mn-PGHS-1 [(A) 18.5 and (B) 20 µM] in 100 mM KP_i (pH 7.2), 0.04% octyl glucoside, and 10% glycerol was reacted at room temperature with either 5 (A) or 2 equiv (B) of 15-HPETE. Tyrosyl radical levels were determined by double integration of EPR signals such as those shown in Figure 3 (●); Mn⁵⁺=O concentrations are reflected by A₄₂₀ in parallel stopped-flow experiments (─). Dashed lines indicate exponential fits for tyrosyl radical formation and decay phases.

FIGURE 6

Comparison of tyrosyl radical, oxyferryl heme, and peroxidase inactivation kinetics of Fe-PGHS-1 during reaction with EtOOH. Fe-PGHS-1 (30 µM) in 100 mM KP_i (pH 7.2), 0.04% octyl glucoside, and 10% glycerol was reacted with 20 equiv of EtOOH at room temperature. (A) Time courses of residual peroxidase activity (♦; fit,─), overall tyrosyl radical intensity (○; fit, ─ ─ ─), deconvoluted WD tyrosyl radical intensity (▲; fit, •••), and oxyferryl heme (─••─). (B) EPR spectra from reaction samples freeze-trapped at the indicated times. EPR conditions: microwave power, 1 mW; frequency, 9.297 GHz; modulation amplitude, 2 G; and temperature, 114 K.

FIGURE 7

Comparison of tyrosyl radical, oxyferryl heme, and peroxidase inactivation kinetics of Fe-PGHS-1 during reaction with 15-HPETE. Fe-PGHS-1 (30 µM) in 100 mM KP_i (pH 7.2), 0.04% octyl glucoside, and 10% glycerol was reacted with 20 equiv of 15-HPETE at room temperature. (A) Time course of residual peroxidase activity (♦; fit, ─), tyrosyl radical intensity (○; fit, ─ ─ ─), deconvoluted WD concentration (▲; fit, •••), and oxyferryl heme (─•─). (B) EPR spectra from reaction samples freeze-trapped at the indicated times. EPR conditions were the same as those described in the legend of Figure 6.

FIGURE 8

Comparison of tyrosyl radical, oxyferryl heme, and peroxidase inactivation kinetics of indomethacin-treated Fe-PGHS-1 during reaction with 15-HPETE. Indo-Fe-PGHS-1 (30 µM) in 100 mM KP_i (pH 7.2), 0.04% octyl glucoside, and 10% glycerol was reacted with 20 equiv of 15-HPETE at room temperature. (A) Time course of residual peroxidase activity (♦; fit, ─), tyrosyl radical intensity (○; fit, ─ ─ ─), and oxyferryl heme (─•─). (B) EPR spectra from reaction samples freeze-trapped at the indicated times. EPR conditions were the same as those described in the legend of Figure 6.

FIGURE 9

Comparison of observed kinetics of A₄₂₈ (●) and normalized remaining peroxidase activity (□) during reaction of Fe-PGHS-1 with 20 equiv of EtOOH (A) or 15-HPETE (B) from the experiments in Figure 6 and Figure 7 with computer simulations (─) generated as described in Experimental Procedures. The optimal values of rate constants for reaction with EtOOH are listed in Table 1, and the following parameters were used: ε1 = 0.063 µM⁻¹ cm⁻¹, ε2 = 0.102 µM⁻¹ cm⁻¹, ε3 = 0.080 µM⁻¹ cm⁻¹, ε4 = 0.061 µM⁻¹ cm⁻¹, and Residual = 0.34. The optimal values of rate constants for reaction with 15-HPETE are listed in Table 1, and the following parameters were used: ε1 = 0.060 µM⁻¹ cm⁻¹, ε2 = 0.091 µM⁻¹ cm⁻¹, ε3 = 0.068 µM⁻¹ cm⁻¹, ε4 = 0.049 µM⁻¹ cm⁻¹, and Residual = 0.05.