Peroxide-induced radical formation at TYR385 and TYR504 in human PGHS-1

Abstract

Prostaglandin H synthase isoforms 1 and -2 (PGHS-1 and -2) react with peroxide to form a radical on Tyr385 that initiates the cyclooxygenase catalysis. The tyrosyl radical EPR signals of PGHS-1 and -2 change over time and are altered by cyclooxygenase inhibitor binding. We characterized the tyrosyl radical dynamics using wild type human PGHS-1 (hPGHS-1) and its Y504F, Y385F, and Y385F/Y504F mutants to determine whether the radical EPR signal changes involve Tyr504 radical formation, Tyr385 radical phenyl ring rotation, or both. Reaction of hPGHS-1 with peroxide produced a wide singlet, whereas its Y504F mutant produced only a wide doublet signal, assigned to the Tyr385 radical. The cyclooxygenase specific activity and K(M) value for arachidonate of hPGHS-1 were not affected by the Y504F mutation, but the peroxidase specific activity and the K(M) value for peroxide were increased. The Y385F and Y385F/Y504F mutants retained only a small fraction of the peroxidase activity; the former had a much-reduced yield of peroxide-induced radical and the latter essentially none. After binding of indomethacin, a cyclooxygenase inhibitor, hPGHS-1 produced a narrow singlet but the Y504F mutant did not form a tyrosyl radical. These results indicate that peroxide-induced radicals form on Tyr385 and Tyr504 of hPGHS-1, with radical primarily on Tyr504 in the wild type protein; indomethacin binding prevented radical formation on Tyr385 but allowed radical formation on Tyr504. Thus, hPGHS-1 and -2 have different distributions of peroxide-derived radical between Tyr385 and Tyr504. Y504F mutants in both hPGHS-1 and -2 significantly decreased the cyclooxygenase activation efficiency, indicating that formation of the Tyr504 radical is functionally important for both isoforms.

Figure 1

Depiction of oPGHS-1 structure near the peroxidase pocket (PDB entry 2AYL) showing Tyr385 (red stick), Tyr504 (blue stick), Mn-PPIX (green stick), the proximal ligand, His388 (orange stick), His207 (yellow stick) [64]. The red sphere is a structural water H-bonded to Tyr504 and His388.

Figure 2

Radicals EPR signals during reaction of hPGHS-1 with EtOOH. (A) hPGHS-1 (47 μM heme) in 100 mM KPi, pH 7.2, 0.1% Tween-20, 10% glycerol was reacted at room temperature with 5 equiv of EtOOH for the indicated times before the sample was freeze-quenched and the EPR spectrum recorded. Vertical dashed lines indicate the peak and trough positions in the 4 ms spectrum. (B) The radical intensity in each sample was determined by double integration and normalized to the hPGHS-1 heme concentration. The line represents the first-order fit to the data.

Figure 3

EPR spectra of radicals formed upon reaction of wild type or mutant hPGHS-1 with EtOOH. The samples (21 μM heme) in 100 mM KPi, pH 7.2, 0.1% Tween-20, 10% glycerol were reacted on ice with 5 equiv of EtOOH for ~10 s. The range of radical intensity (in spin/heme) obtained in replicate experiments is indicated at the left side of each spectrum.

Figure 4

EPR spectra of radicals formed by indomethacin complexes of wild type and mutant hPGHS-1 upon reaction with EtOOH. Each sample (21 μM heme) in 100 mM KPi, pH 7.2, 0.1% Tween-20, was preincubated with 2 equiv of indomethacin before reaction on ice with 5 equiv of EtOOH for ~10 s. The range of radical intensity (in spin/heme) obtained in replicate experiments is indicated at the left side of each spectrum.

Figure 5

Tyrosyl radical EPR spectra during reaction of the meclofenamate complex of hPGHS-1 with EtOOH. hPGHS-1 (29 μM heme) pre-treated with 2 equivalents of meclofenamate in 100 mM KPi, pH 7.2, 0.1% Tween-20, was reacted on ice with 5 equiv of EtOOH for ~10 s, frozen, and the first EPR spectrum was recorded. For recording subsequent spectra, the sample was thawed and allowed to react at 0 °C for ~10 s before being re-frozen. The arrow indicates the direction of amplitude changes.

Figure 6

Simulation of peroxide-induced tyrosyl radical EPR signals from wild type and mutant hPGHS-1. (A) Experimental (––––) and simulated (– • –) EPR spectra of the wide doublet (WD; Y504F hPGHS-1, 9 s at 0 °C), narrow singlet (NS; indomethacin-hPGHS-1, 9 s at 0 °C), and early wide singlet (WS1; hPGHS-1, 4 ms at room temperature) EPR signals. Parameters for the simulations are given in Table 3. The S.E.E. values for the agreement between observed and simulated spectra are indicated at the left side. (B) Structural diagrams of the proposed conformations of the beta-methylene groups of tyrosyl radicals for the simulated spectra.

Figure 7

Structure of the vicinity of Tyr504 in oPGHS-1 showing Ile440, Gln383, Met525, His388 and Mn-PPIX (PDB entry 2AYL: Gupta K et al. (2006) Acta Cryst D 62, 151). The corresponding amino acid residues in hPGHS-1 are indicated after the position number.