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Dendritic cells (DCs) are important mediators of the induction and regulation of adaptive immune responses following microbial infection and inflammation. Sensing environmental danger signals including viruses, microbial products, or inflammatory stimuli by DCs leads to the rapid transition from a resting state to an activated mature state. DC maturation involves enhanced capturing and processing of antigens for presentation by major histocompatibility complex (MHC) class I and class II, upregulation of chemokines and their receptors, cytokines and costimulatory molecules, and migration to lymphoid tissues where they prime naive T cells. Orchestrating a cellular response to environmental threats requires a high bioenergetic cost that accompanies the metabolic reprogramming of DCs during activation. We previously demonstrated that DCs undergo a striking functional transition after stimulation of the retinoic acid-inducible gene I (RIG-I) pathway with a synthetic 5' triphosphate containing RNA (termed M8), consisting of the upregulation of interferon (IFN)-stimulated antiviral genes, increased DC phagocytosis, activation of a proinflammatory phenotype, and induction of markers associated with immunogenic cell death. In the present study, we set out to determine the metabolic changes associated with RIG-I stimulation by M8. The rate of glycolysis in primary human DCs was increased in response to RIG-I activation, and glycolytic reprogramming was an essential requirement for DC activation. Pharmacological inhibition of glycolysis in monocyte-derived dendritic cells (MoDCs) impaired type I IFN induction and signaling by disrupting the TBK1-IRF3-STAT1 axis, thereby countering the antiviral activity induced by M8. Functionally, the impaired IFN response resulted in enhanced viral replication of dengue, coronavirus 229E, and Coxsackie B5., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zevini, Palermo, Di Carlo, Alexandridi, Rinaldo, Paone, Cutruzzola, Etna, Coccia, Olagnier and Hiscott.)

Among the over 50 gyrate atrophy-causing mutations of ornithine δ-aminotransferase (OAT), the R180T involves an active site residue located at the dimer interface, which in the crystal structure of OAT complexed with 5-fluoromethylornithine engages a salt bridge with the α-carboxylate of the substrate analogue. Starting from the previous finding that no transaminase activity was detected in CHO-K 1 cells expressing the R180T variant, here we try to shed light at the protein level on the structural and/or functional defects of the R180T variant. To this aim, the variant has been cloned, expressed, purified and characterized by a combination of biochemical and structural studies. Although the R180T variant shares a similar overall conformation with the wild-type, its crystal structure solved at 1.8 Ǻ reveals slight structural alterations at the active site and at the dimeric interface. These changes are consistent with the spectroscopic and kinetic results, indicating that the variant, as compared with the wild-type OAT, shows (a) an increased K m value for l-ornithine (l-Orn), (b) an altered pyridoxal 5'-phosphate binding mode and affinity and (c) an increased thermostability. In addition, the R180T mutant exhibits a remarkable loss of catalytic activity and is endowed with the ability to catalyse not only the δ-transamination but also, albeit to a lesser extent, the α-transamination of l-Orn. Overall, these data indicate that the slight structural changes caused by the R180T mutation, preventing a proper collocation of l-Orn at the active site of OAT, are responsible for the notable reduction of the catalytic efficiency. ENZYMES: Ornithine aminotransferase EC 2.6.1.13. DATABASES: 6HX7.pdb., (© 2019 Federation of European Biochemical Societies.)

A wide range of organic reductants, including many iron chelators, reduce ferryl myoglobin to its ferric states in exponential time courses whose rate constants display double hyperbolic dependencies on the reductant concentration. This concentration dependence is consistent with a mechanism in which electron transfer to the heme takes place at two independent sites where reductants appear to bind. We propose that the low-affinity site is located close to the heme edge, within the heme pocket; the maximum rate of electron transfer is highly variable depending on the nature of the reductant (0.005 to >10 s(-1)). The other site has higher apparent affinity (K(D) 0.2-50 microM) but a low maximum rate of electron transfer (0.005 to 0.01 s(-1)). By examining native and engineered proteins we have determined that the high-affinity pathway represents a through-protein electron transfer pathway that involves a specific tyrosine residue. The low apparent rate constant for electron transfer from the tyrosine to the heme (approximately 5 A) is accounted for by proposing that electron transfer occurs only in a very poorly populated protonated state of ferryl heme and tyrosine. Hemoglobin shows similar kinetics but only one subunit exhibits double rectangular hyperbolic concentration dependency. The consequence of a high-affinity through-protein electron transfer pathway to the cytotoxicity of ferryl heme is discussed.

The nitrite reductase (NIR) from Pseudomonas aeruginosa (NIR-Pa) is a soluble enzyme catalysing the reduction of nitrite (NO2(-)) to nitric oxide (NO). The enzyme is a 120 kDa homodimer, in which the monomers carry a c-heme domain and a d(1)-heme domain. The structures of the enzyme in both the oxidised and reduced state were solved previously and indicate His327 and His369 as putative catalytic residues. The kinetic characterisation of site-directed mutants has shown that the substitution of either one of these two His with Ala dramatically reduces the physiologically relevant reactivity towards nitrite, leaving the reactivity towards oxygen unaffected. The three-dimensional structures of P. aeruginosa NIR mutant H327A, and H369A in complex with NO have been solved by multiple wavelength anomalous dispersion (MAD), using the iron anomalous signal, and molecular replacement techniques. In both refined crystal structures the c-heme domain, whilst preserving its classical c-type cytochrome fold, has undergone a 60 degrees rigid-body rotation around an axis parallel with the pseudo 8-fold axis of the beta-propeller, and passing through residue Gln115. Even though the distance between the Fe ions of the c and d(1)-heme remains 21 A, the edge-to-edge distance between the two hemes has increased by 5 A. Furthermore the distal side of the d(1)-heme pocket appears to have undergone structural re-arrangement and Tyr10 has moved out of the active site. In the H369A-NO complex, the position and orientation of NO is significantly different from that of the NO bound to the reduced wild-type structure. Our results provide insight into the flexibility of the enzyme and the distinction between nitrite and oxidase reduction mechanisms. Moreover they demonstrate that the two histidine residues play a crucial role in the physiological activity of nitrite reduction, ligand binding and in the structural organisation of nitrite reductase from P. aeruginosa., (Copyright 2001 Academic Press.)

Cd(1) nitrite reductase catalyzes the conversion of nitrite to NO in denitrifying bacteria. Reduction of the substrate occurs at the d(1)-heme site, which faces on the distal side some residues thought to be essential for substrate binding and catalysis. We report the results obtained by mutating to Ala the two invariant active site histidines, His-327 and His-369, of the enzyme from Pseudomonas aeruginosa. Both mutants have lost nitrite reductase activity but maintain the ability to reduce O(2) to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form, possibly because the productive displacement of NO from the ferric d(1)-heme iron is impaired. Moreover, the two distal His play different roles in catalysis; His-369 is absolutely essential for the stability of the Michaelis complex. The structures of both mutants show (i) the new side chain in the active site, (ii) a loss of density of Tyr-10, which slipped away with the N-terminal arm, and (iii) a large topological change in the whole c-heme domain, which is displaced 20 A from the position occupied in the wild-type enzyme. We conclude that the two invariant His play a crucial role in the activity and the structural organization of cd(1) nitrite reductase from P. aeruginosa.