*4.3. Lipoxygenase Assay*

Human recombinant 15LOX-2 and PEBP1 were recombinantly expressed an purified as described previously [3,63]. 15LOX-2 was pre-activated with 13-hydroperoxyoctadecadienoic acid (HpODE) (5 μM) for 30 min at 37 ◦C. The pre-activated 15LOX-2 (3 pmols) was added to a reaction mixture containing 100 μM lipid in Tris-HCl (50 mM, pH7.4), 25 μM PAPA NONOAte and DTPA (100 μM) for a total volume of 50 μL. DTPA was added 15 min prior to the start of the reaction. For reactions with the 15LOX-2/PEBP1 complex, equal quantity of PEBP1 and 15LOX-2 were mixed prior to pre-activation. The reaction mixture was incubated on a shaker mixer at 37 ◦C for 30 min. For time course, the reaction was allowed to continue up to a specific time. The reaction was stopped with addition of 9 volumes (450 μL) of 100% acetonitrile, and the samples were centrifuged at 10,000× *g* for 15 min at 4 ◦C. Then, 20 μL of the supernatant was transferred to an auto sampler vial, and 5 μL was injected into the LC-MS/MS system. LC-MS/MS analysis was performed as described previously.

## *4.4. Cell Culture*

RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC). Cultured at 37 ◦C and 5% CO2 in DMEM or RPMI (ATCC) supplemented with 10% heat-inactivated fetal calf serum (FCS; Sigma–Aldrich, St. Louis, MO, USA) and 50 U ml−<sup>1</sup> penicillin–streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). RAW 264.7 macrophages were polarized by to M2 stage by incubating in DMEM containing 10% FBS, 50 U mL−<sup>1</sup> penicillin–streptomycin, and IL-4 (20 ng mL−1) for 48 h. For ferroptotis experiments, cells were incubated with RSL3 (0.5 μM), 2 μM), +Fer-1 (~400 nM), or ±DPTA NONOate (25 μM), for 5 h. Cell death was determined by flow cytometry.

#### *4.5. LC–MS Analysis of Phospholipids*

MS analysis of phospholipids was performed on an Orbitrap mass spectrometer (Thermo Fisher). Phospholipids were separated on a normal phase column (Luna 3 μm Silica (2) 100 Å, 150 × 2.0 mm, Phenomenex, Torrance, CA, USA) at a flow rate of 0.2 mL min−<sup>1</sup> on a Dionex Ultimate 3000 HPLC system (Dionex, Idstein, Germany). The column was maintained at 35 ◦C. Analysis was performed using gradient solvents (A and B) containing 10 mM ammonium acetate. Solvent A contained propanol:hexane:water (285:215:5, vol/vol/vol) and solvent B contained propanol:hexane:water (285:215:40, vol/vol/vol). All solvents were LC–MS grade. The column was eluted for 0–23 min with a linear gradient of 10–32% B; 23–32 min using a linear gradient of 32–65% B; 32–35 min with a linear gradient of 65–100% B; 35–62 min held at 100% B; 62–64 min with a linear gradient from 100% to 10% B followed by and equilibration from 64 to 80 min at 10% B. The instrument was operated with the electrospray ionization probe in negative polarity mode. Data was analyzed using Quan Browser of xcalibur software (Thermo Fisher).

**Supplementary Materials:** The following are available online at https://www.mdpi.com/1422-0 067/22/10/5253/s1, Figure S1: Location of the two entrances E1 and E2 of 15LOX-2 that enable access of O2 and NO• to the catalytic site, Figure S2: Secondary structure of 15LOX-2, Figure S3: Additional pores or tunnels leading to the catalytic site, Figure S4: Close-up view of contacts between 15LOX-2 residues and O2/NO• molecules, Figure S5: Same results as Figure 4a–b, reproduced by an independent run, Figure S6: 15LOX-2/SAPE complex and its interactions with O2 and NO• molecules observed in MD simulations with higher concentration of NO• molecules, Figure S7: 15LOX-2/AA complex and its interactions with O2 and NO• molecules observed in MD simulations, Table S1: Summary of the simulated systems, compositions, and durations, Table S2: Key residues in 15LOX-2 that play a role in regulation of lipid peroxidation, Movie 1: Competition between NO• and O2 molecules near the SAPE at the catalytic site of 15LOX-2 complexed with PEBP1.

**Author Contributions:** V.E.K., K.M.-R., and I.B. conceived the study. T.S.A. designed lipidomics experiments. V.E.K. and H.B. designed experiments; T.S.A. and A.L. performed lipidomics experiments and analyzed the experimental data. A.A.K. performed RAW M2 macrophages experiments. K.M.-R. and I.H.S. designed the computational experiments. K.M.-R. performed the computational modeling and simulations. I.B., K.M.-R., and I.H.S. interpreted the MD data. K.M.-R., I.B., and V.E.K. wrote the manuscript, which was edited by all authors. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by NIH (HL114453, U01AI156924, U01AI156923, CA165065, NS076511, NS061817, P41 GM103712, P01 DK096990), and by Polish National Science Centre no. 2019/35/D/ST4/02203.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data and codes generated during the study and included in this article are available from the corresponding authors upon request.

**Acknowledgments:** This work was supported by NIH (HL114453, U01AI156924, U01AI156923, CA165065, NS076511, NS061817, P41 GM103712, P01 DK096990), by Polish National Science Centre no. 2019/35/D/ST4/02203. Karolina Mikulska-Ruminska is thankful for the facilities and computer time allocated by the Interdisciplinary Center for Modern Technologies, NCU. The authors thank Ted Holman (University of California at Santa Cruz) and Andrew VanDemark (University of Pittsburgh) for their generous gift of recombinant 15LOX-2 and PEBP1, respectively.

**Conflicts of Interest:** The authors have declared that no conflicts of interest exists.
