**1. Introduction**

Recent years have brought attention to ferroptosis, an iron-dependent form of regulated cell death implicated in a broad range of diseases [1,2], and to the selective peroxidation of eicosatetraenoyl-phosphatidylethanolamine (ETE-PE) by 15-lipoxygenase (15LOX) complexed with PE-binding protein 1 (PEBP1) [3–5] as a ferroptotic mechanism. ETE-PE

**Citation:** Mikulska-Ruminska, K.; Anthonymuthu, T.S.; Levkina, A.; Shrivastava, I.H.; Kapralov, A.A.; Bayır, H.; Kagan, V.E.; Bahar, I. NO• Represses the Oxygenation of Arachidonoyl PE by 15LOX/PEBP1: Mechanism and Role in Ferroptosis. *Int. J. Mol. Sci.* **2021**, *22*, 5253. https://doi.org/10.3390/ijms22105253 **\***

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Academic Editor: Masoud Jelokhani-Niaraki

Received: 3 April 2021 Accepted: 12 May 2021 Published: 17 May 2021

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peroxidation produces hydroperoxy-ETE-PE (HpETE-PE) molecules that, in turn, serve as pro-ferroptotic signals. 15LOX catalytic action includes the Fe-driven abstraction of a hydrogen atom from the carbon in bis-allylic position, formation of a carbon-centered radical followed by addition of molecular oxygen, culminating in the production of 15-HpETE.

The role of 15LOX in the onset of ferroptosis is now well-established [3–6]. Under physiological conditions, arachidonic acid (AA), a polyunsaturated fatty acid (PUFA) also termed *cis*-5,8,11,14-ETE or shortly ETE, is the preferred substrate of these non-heme iron-containing enzymes; but under inflammatory conditions, such as those stimulated by interleukin-13/4, the formation of the complex 15LOX/PEBP1 shifts the substratespecificity of 15LOX from free AA to AA esterified into PE (or ETE-PE). The AA-esterification of PE mostly occurs at the sn-2 acyl chain leaving the sn-1 chain for saturated or monounsaturated acyls, predominantly palmitic (C16:0), stearic (S) (C18:0), and oleic (C18:1). Of interest below will be 1-SA-2-ETE-PE, i.e., PE with stearic acid (SA) and arachidonic acid (AA or ETE) at the respective sn-1 and sn-2 chains, which will be shortly called SAPE. 15LOX-catalyzed peroxidation of SAPE at its sn-2 chain results in the production of 15- HpHETE-PE [3]—one of the major players in the ensuing induction of ferroptosis. As such, the 15LOX-2/PEBP1 complex emerged as a master promoter of ferroptotic cell-death [3]. This involvement of 15LOX-2/PEBP1 has been supported by the ability of ferrostatin-1, the most common ferroptosis inhibitor, to bind the complex [4] and inhibit the formation of 15-HpHETE-PE.

As a multistage process, ferroptosis is regulated by several enzymatic events occurring independently or concertedly: (i) biosynthesis of PUFA-PE peroxidation substrates by acyl Co-A synthetase 4 (ACSL4) and lysophosphatidylcholine acyltransferase (LPCAT) [7]; (ii) prevention of the formation of 15-HpETE-PE by flavin-containing oxidoreductase, ferroptosis suppressor protein 1 (FSP1) [8,9]; and (iii) reduction in 15-HpETE-PE into an innocuous alcohol, 15-HETE-PE, by glutathione peroxidase 4 (GPX4) [10,11]. Lately, this list of ferroptosis regulators has been extended to include new members, such as the Ca2+-independent phospholipase A2β (iPLA2β) [12], which can hydrolyze peroxidized phospholipids and, therefore, can eliminate the ferroptotic cell death signal, and the inducible isoform of nitric oxide synthase, iNOS, that generates NO• [13]. Notably, NO•'s reactivity towards different radical intermediates of lipid peroxidation has been discovered more than three decades ago [14]. Furthermore, NO• inhibits lipid peroxidation by lipoxygenase and cyclooxygenase [15].

In the present study, we first performed a lipidomics analysis to observe that NO• indeed suppresses the production of HpETE-PE by 15LOX-2/PEBP1, while promoting the formation of nitrosylated ETE-PE. As the 15LOX-2/PEBP1 complex, rather than 15LOX-2 alone, catalyzes the oxidation of 15-ETE-PE (rather than free ETE), we focused here on the interactions of NO• with SAPE and15LOX-2/PEBP1, and on its competition with the lipoxygenase substrate O2. Notably, the precise delivery of molecular oxygen to form a peroxyl radical from the carbon-centered radical of ETE at the 15LOX-2 catalytic site occurs via a specialized channel, whose structure and accessibility is altered upon binding of ETE to the enzyme [16]. With this in mind, we studied the effects of NO• peroxidation of SAPE by 15LOX-2/PEBP1 and the structural dynamics of the oxygen delivery process to the catalytic Fe3+ in the 15LOX-2/PEBP1 complex and the competition with NO•, in the presence and absence of the ETE-PE substrate compared to free ETE in 15LOX-2. Our study provides a mechanistic description of the time-evolved interactions between 15LOX-2 (bound to PEBP1) and its ligands, the ETE-PE- and PEBP1-induced changes in 15LOX-2 conformation or catalytic site accessibility which alter the delivery of O2 and NO•, and the competition between O2 and NO• for binding the active site. Several key residues mediating the events prior to peroxidation and the interference of NO• to repress them are identified, the critical roles of which are consolidated by in silico saturation mutagenesis experiments and comparisons with recent work.
