Nuclear Resonance Vibrational Spectroscopy Definition of Peroxy Intermediates in Catechol Dioxygenases: Factors that Determine Extra- versus Intradiol Cleavage.

The extradiol dioxygenases (EDOs) and intradiol dioxygenases (IDOs) are nonheme iron enzymes that catalyze the oxidative aromatic ring cleavage of catechol substrates, playing an essential role in the carbon cycle. The EDOs and IDOs utilize very different FeII and FeIII active sites to catalyze the regiospecificity in their catechol ring cleavage products. The factors governing this difference in cleavage have remained undefined. The EDO homoprotocatechuate 2,3-dioxygenase (HPCD) and IDO protocatechuate 3,4-dioxygenase (PCD) provide an opportunity to understand this selectivity, as key O2 intermediates have been trapped for both enzymes. Nuclear resonance vibrational spectroscopy (in conjunction with density functional theory calculations) is used to define the geometric and electronic structures of these intermediates as FeII-alkylhydroperoxo (HPCD) and FeIII-alkylperoxo (PCD) species. Critically, in both intermediates, the initial peroxo bond orientation is directed toward extradiol product formation. Reaction coordinate calculations were thus performed to evaluate both the extra- and intradiol O-O cleavage for the simple organic alkylhydroperoxo and for the FeII and FeIII metal catalyzed reactions. These results show the FeII-alkylhydroperoxo (EDO) intermediate undergoes facile extradiol O-O bond homolysis due to its extra e-, while for the FeIII-alkylperoxo (IDO) intermediate the extradiol cleavage involves a large barrier and would yield the incorrect extradiol product. This prompted our evaluation of a viable mechanism to rearrange the FeIII-alkylperoxo IDO intermediate for intradiol cleavage, revealing a key role in the rebinding of the displaced Tyr447 ligand in this rearrangement, driven by the proton delivery necessary for O-O bond cleavage.

Mechanisms of O2 Activation by Mononuclear Non-Heme Iron Enzymes.

Two major subclasses of mononuclear non-heme ferrous enzymes use two electron-donating organic cofactors (α-ketoglutarate or pterin) to activate O2 to form FeIV═O intermediates that further react with their substrates through hydrogen atom abstraction or electrophilic aromatic substitution. New spectroscopic methodologies have been developed, enabling the study of the active sites in these enzymes and their oxygen intermediates. Coupled to electronic structure calculations, the results of these spectroscopies provide fundamental insight into mechanism. This Perspective summarizes the results of these studies in elucidating the mechanism of dioxygen activation to form the FeIV═O intermediate and the geometric and electronic structure of this intermediate that enables its high reactivity and selectivity in product formation.

Macrophage phenotype and bioenergetics are controlled by oxidized phospholipids identified in lean and obese adipose tissue.

Adipose tissue macrophages (ATMs) adapt their metabolic phenotype either to maintain lean tissue homeostasis or drive inflammation and insulin resistance in obesity. However, the factors in the adipose tissue microenvironment that control ATM phenotypic polarization and bioenergetics remain unknown. We have recently shown that oxidized phospholipids (OxPL) uniquely regulate gene expression and cellular metabolism in Mox macrophages, but the presence of the Mox phenotype in adipose tissue has not been reported. Here we show, using extracellular flux analysis, that ATMs isolated from lean mice are metabolically inhibited. We identify a unique population of CX3CR1neg/F4/80low ATMs that resemble the Mox (Txnrd1+HO1+) phenotype to be the predominant ATM phenotype in lean adipose tissue. In contrast, ATMs isolated from obese mice had characteristics typical of the M1/M2 (CD11c+CD206+) phenotype with highly activated bioenergetics. Quantifying individual OxPL species in the stromal vascular fraction of murine adipose tissue, using targeted liquid chromatography-mass spectrometry, revealed that high fat diet-induced adipose tissue expansion led to a disproportional increase in full-length over truncated OxPL species. In vitro studies showed that macrophages respond to truncated OxPL species by suppressing bioenergetics and up-regulating antioxidant programs, mimicking the Mox phenotype of ATMs isolated from lean mice. Conversely, full-length OxPL species induce proinflammatory gene expression and an activated bioenergetic profile that mimics ATMs isolated from obese mice. Together, these data identify a redox-regulatory Mox macrophage phenotype to be predominant in lean adipose tissue and demonstrate that individual OxPL species that accumulate in adipose tissue instruct ATMs to adapt their phenotype and bioenergetic profile to either maintain redox homeostasis or to promote inflammation.

Macrophages sensing oxidized DAMPs reprogram their metabolism to support redox homeostasis and inflammation through a TLR2-Syk-ceramide dependent mechanism.

OBJECTIVE: Macrophages control tissue homeostasis and inflammation by sensing and responding to environmental cues. However, the metabolic adaptation of macrophages to oxidative tissue damage and its translation into inflammatory mechanisms remains enigmatic. METHODS: Here we identify the critical regulatory pathways that are induced by endogenous oxidation-derived DAMPs (oxidized phospholipids, OxPL) in vitro, leading to formation of a unique redox-regulatory metabolic phenotype (Mox), which is strikingly different from conventional classical or alternative macrophage activation. RESULTS: Unexpectedly, metabolomic analyses demonstrated that Mox heavily rely on glucose metabolism and the pentose phosphate pathway (PPP) to support GSH production and Nrf2-dependent antioxidant gene expression. While the metabolic adaptation of macrophages to OxPL involved transient suppression of aerobic glycolysis, it also led to upregulation of inflammatory gene expression. In contrast to classically activated (M1) macrophages, Hif1α mediated expression of OxPL-induced Glut1 and VEGF but was dispensable for Il1ß expression. Mechanistically, we show that OxPL suppress mitochondrial respiration via TLR2-dependent ceramide production, redirecting TCA metabolites to GSH synthesis. Finally, we identify spleen tyrosine kinase (Syk) as a critical downstream signaling mediator that translates OxPL-induced effects into ceramide production and inflammatory gene regulation. CONCLUSIONS: Together, these data demonstrate the metabolic and bioenergetic requirements that enable macrophages to translate tissue oxidation status into either antioxidant or inflammatory responses via sensing OxPL. Targeting dysregulated redox homeostasis in macrophages could therefore lead to novel therapies to treat chronic inflammation.