Multifrequency Electron Paramagnetic Resonance Characterization of PpoA, a CYP450 Fusion Protein that Catalyzes Fatty Acid Dioxygenation

Abstract

PpoA is a fungal dioxygenase that produces hydroxylated fatty acids involved in the regulation of the life cycle and secondary metabolism of Aspergillus nidulans. It was recently proposed that this novel enzyme employs two different heme domains to catalyze two separate reactions: within a heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid [(8R)-HPODE]; in the second reaction step (8R)-HPODE is isomerized within a P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. In the present study, pulsed EPR methods were applied to find spectroscopic evidence for the reaction mechanism, thought to involve paramagnetic intermediates. We observe EPR resonances of two distinct heme centers with g-values typical for Fe(III) S = 5/2 high-spin (HS) and Fe(III) S = 1/2 low-spin (LS) hemes. 14N ENDOR spectroscopy on the S = 5/2 signal reveals resonances consistent with an axial histidine ligation. Reaction of PpoA with the substrate leads to the formation of an amino acid radical on the early millisecond time scale concomitant to a substantial reduction of the S = 5/2 heme signal. High-frequency EPR (95- and 180-GHz) unambiguously identifies the new radical as a tyrosyl, based on g-values and hyperfine couplings from spectral simulations. The radical displays enhanced T 1-spin–lattice relaxation due to the proximity of the heme centers. Further, EPR distance measurements revealed that the radical is distributed among the monomeric subunits of the tetrameric enzyme at a distance of approximately 5 nm. The identification of three active paramagnetic centers involved in the reaction of PpoA supports the previously proposed reaction mechanism based on radical chemistry.

Production of cross-kingdom oxylipins by pathogenic fungi: An update on their role in development and pathogenicity

“…The N-terminal heme peroxidase domain predominantly oxidizes linoleic acid to 8(R)-hydroperoxyoctadecadienoic acid (8(R)-HPODE) which can be reduced to 8-hydroxyoctadecadienoic acid (8(R)-HODE), or further isomerized by the C-terminal P450 heme thiolate domain to 5,8-dihydroxyoctadecadieonic acid (5,8-DiHODE) (Garscha et al ., 2007; Brodhun, et al ., 2009; Fielding et al ., 2011). Studies with GFP-tagged forms of PpoA reveal that PpoA localizes to lipid bodies within the reproductive structures of A. nidulans , including the metulae of the asexual conidiophore and the Hülle cells and young cleistothecia of the sexual structures.…”

Cross-polarisation edited ENDOR

“…here [i-j] equal [1][2][3] and [2][3][4] for the {e-n} spin system and [1][2][3][4][5], [2][3][4][5][6], [3][4][5][6][7] and [4][5][6][7][8] for the {e-n 1 -n 2 } spin system. Angle ϕ [i−j ] corresponds to the projection of the effective field in the [i-j] electron manifold on the x-y plane.…”

“…Here, not only orientational selectivity but also increased resolution in the nuclear Larmor frequencies and suppression of second-order effects substantially simplify analysis and interpretation of hyperfine spectra. Representative examples for the application of ENDOR have been the elucidation of the ligand sphere of metal centres or the detection of hydrogen bond interactions in proteins [7][8][9][10]. Nevertheless, one substantial drawback of the currently used ENDOR techniques is the small ENDOR effect as compared to the available EPR signal.…”

Stereoselective oxidation of regioisomeric octadecenoic acids by fatty acid dioxygenases

“…COX-1 and the oleate 8 R -DOX activities of 7,8-LDS, 5,8-LDS, and 10 R -DOX initiate oxidation of octadecenoic acids by hydrogen abstraction, which likely is catalyzed by a tyrosyl radical ( 16,40,41 ). The selective oxidation of three octadecenoic acids by ovine COX-1 proved that C-11 of (12 Z ) -18:1, C-12 of (13 Z ) -18:1, and both C-10 and C-13 of (11 Z ) -18:1 were positioned suffi ciently close to Tyr-385 for hydrogen abstraction.…”