Structural comparisons of arachidonic acid-induced radicals formed by prostaglandin H synthase-1 and -2

Abstract

Cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2 involves reaction of a peroxide-induced Tyr385 radical with arachidonic acid (AA) to form an AA radical that reacts with O(2). The potential for isomeric AA radicals and formation of an alternate tyrosyl radical at Tyr504 complicate analysis of radical intermediates. We compared the EPR spectra of PGHS-1 and -2 reacted with peroxide and AA or specifically deuterated AA in anaerobic, single-turnover experiments. With peroxide-treated PGHS-2, the carbon-centered radical observed after AA addition was consistently a pentadienyl radical; a variable wide-singlet (WS) contribution from mixture of Tyr385 and Tyr504 radicals was also present. Analogous reactions with PGHS-1 produced EPR signals consistent with varying proportions of pentadienyl and tyrosyl radicals, and two additional EPR signals. One, insensitive to oxygen exposure, is the narrow singlet tyrosyl radical with clear hyperfine features found previously in inhibitor-pretreated PGHS-1. The second type of EPR signal is a narrow singlet lacking detailed hyperfine features that disappeared upon oxygen exposure. This signal was previously ascribed to an allyl radical, but high field EPR analysis indicated that ~90% of the signal originates from a novel tyrosyl radical, with a small contribution from a carbon-centered species. The radical kinetics could be resolved by global analysis of EPR spectra of samples trapped at various times during anaerobic reaction of PGHS-1 with a mixture of peroxide and AA. The improved understanding of the dynamics of AA and tyrosyl radicals in PGHS-1 and -2 will be useful for elucidating details of the cyclooxygenase mechanism, particularly the H-transfer between tyrosyl radical and AA.

Fig. 1

EPR spectra observed during anaerobic reaction of PGHS-1 with peroxide and AA. PGHS-1 (0.1mM) in 0.1 M KPi, pH 7.3, containing 15% glycerol and 0.1% Tween-20 was mixed at 23 °C with an equal volume of 0.1 MTris, pH 8.0 containing EtOOH (100 µM), AA (100 µM) and 0.05% Tween-20. Samples were freeze-trapped after the indicated times at 117–123 K and the EPR spectra were recorded. The spectrometer conditions were: microwave frequency, 9.29 GHz; power, 1 mW; modulation, 2 G; time constant, 0.33 s and temperature, 114–118 K. The vertical lines indicate the peak-to-trough line width for the 20ms sample, and the arrows indicate EPR features associated with the pentadienyl AA radical. Overall radical intensities (in spin/heme) are shown on the right side of each spectrum. This experiment is one of three repetitions and all of them show similar results.

Fig. 2

Global analysis of EPR spectra obtained during reaction of PGHS-1 (17 µM) with EtOOH (85 µM). A) Experimental EPR spectra for samples quenched at the indicated reaction times; B) kinetic scheme used for global analysis; C) resolved spectra of WD1 and WS1 obtained from global analysis (——) and the corresponding simulated spectra (······); and D) simulated spectra for the time points in Panel A based on the resolved WD1 and WS1 spectra and their predicted concentrations.

Fig. 3

Global analysis of EPR spectra obtained during the reaction of PGHS-1 (100 µM) with EtOOH (100 µM) and 13, 13-d₂-AA (100 µM). A) Experimental EPR spectra for samples quenched at the indicated reaction times; B) kinetic schemes used for global analysis; C) resolved spectra of WD1 and pentadienyl radical obtained from global analysis (——) and the corresponding simulated spectra (······); and D) simulated spectra for the time points in Panel A based on the resolved spectra and their predicted concentrations. The optimized rate constants for mechanism (b) were: k₁=2.8 × 10⁵ M⁻¹ s⁻¹; k₂=0.5 s⁻¹; k₃=0.1 s⁻¹; k₄=0.7 s⁻¹; k₅=0.1 s⁻¹ and k₆=0.002 s⁻¹.

Fig. 4

EPR spectra of radicals generated in sequential reactions at 4 °C of PGHS-1 and -2 with EtOOH and 10,10-d₂-AA. Top: PGHS-1 (15 µM), was reacted with 3 eq of EtOOH for 21 s before mixing with 2 eq of 10,10-d₂-AA and freeze-trapping 11 s later. Center: PGHS-2 (10 µM) was reacted with 10 eq of EtOOH for 21 s before mixing with 4 eq of 10,10-d₂-AA and freeze-trapping 11 s later. Bottom: 5-line AA radical EPR spectrum generated using EtOOH and d₈-AA with the same PGHS-2 preparation under similar conditions. Radical intensities (in spin/heme) are shown at right. Simulated spectra (dashed red lines) for all three samples used the same parameters for the pentadienyl AA radical except that the spectra generated for the 10,10-d₂-AA samples used 1/7 the values for the two β-protons at C10 and I=1.

Fig. 5

HFEPR analysis of radicals in PGHS-1 reacted with AA and EtOOH. A) Solid line: field swept, echo-detected, 130 GHz EPR of PGHS-1 (80 µM) premixed with 5 eq of AA and reacted anaerobically with 10 eq of EtOOH for 5 s at 24 °C before freeze-trapping. Dashed lines: summation of simulated spectra for the tyrosyl and pentadienyl radicals (9:1 ratio) shown in B and C. B: Simulated spectrum for a tyrosyl radical using the following papameters: g_x=2.0070, g_y=2.0042, and g_z=2.0020; full width half height Gaussian distribution of g about g_x=0.003. Principal hyperfine coupling values (in Gauss): two protons (9.5, 2.4, 6.2); two protons (1.0, 4.8, 0.8); one proton (6.7, isotropic); and one proton (7.8, isotropic). The Gaussian line-broadening function was set at 21 G full width half height. C) Simulated spectrum for a pentadienyl radical using parameters determined previously [24]: g_x=2.0032, g_y=2.0032, and g_z=2.0020. Principal hyperfine coupling values (in Gauss, all isotropic): two protons (10.4); two protons (3.3); two protons (16); one proton (11.5); one proton (9.8); and one proton (3.9). The Gaussian line-broadening function was set at 21 G full width half height. The radical concentration for spectrum C is 12% that of spectrum B.

Fig. 6

Cyclooxygenase active site structures for AA bound in a productive orientation to PGHS-1 (panel A; 1DIY [11]) and in reversed orientation to PGHS-2 (panel B; 1CVU [45]). Key residues are labeled and hydrogen bonds are indicated by dashed lines. The phenoxyl oxygen (orange) of Tyr385 (yellow) and the C13 (yellow) of AA (green) involved in H-transfer are highlighted to show the drastic distance change in the two panels.

Scheme 1

Different AA-induced radical EPR signatures found in PGHS-1 and -2 during anaerobic single-turnover manual freeze EPR experiments.