Unlike most other tissues, the colon epithelium is exposed to high levels of H 2 S derived from gut microbial metabolism. H 2 S is a signaling molecule that modulates various physiological effects. It is also a respiratory toxin that inhibits complex IV in the electron transfer chain (ETC). Colon epithelial cells are adapted to high environmental H 2 S exposure as they harbor an efficient mitochondrial H 2 S oxidation pathway, which is dedicated to its disposal. Herein, we report that the sulfide oxidation pathway enzymes are apically localized in human colonic crypts at the host-microbiome interface, but that the normal apical-to-crypt gradient is lost in colorectal cancer epithelium. We found that sulfide quinone oxidoreductase (SQR), which catalyzes the committing step in the mitochondrial sulfide oxidation pathway and couples to complex III, is a critical respiratory shield against H 2 S poisoning. H 2 S at concentrations <20 M stimulated the oxygen consumption rate in colon epithelial cells, but, when SQR expression was ablated, H 2 S concentrations as low as 5 M poisoned cells. Mitochondrial H 2 S oxidation altered cellular bioenergetics, inducing a reductive shift in the NAD ؉ /NADH redox couple. The consequent electron acceptor insufficiency caused uridine and aspartate deficiency and enhanced glutamine-dependent reductive carboxylation. The metabolomic signature of this H 2 S-induced stress response mapped, in part, to redox-sensitive nodes in central carbon metabolism. Colorectal cancer tissues and cell lines appeared to counter the growth-restricting effects of H 2 S by overexpressing sulfide oxidation pathway enzymes. Our findings reveal an alternative mechanism for H 2 S signaling, arising from alterations in mitochondrial bioenergetics that drive metabolic reprogramming

Ergothioneine is a histidine thiol derivative. Its mycobacterial biosynthetic pathway has five steps (EgtA-E catalysis) with two novel reactions: a mononuclear nonheme iron enzyme (EgtB) catalyzed oxidative C–S bond formation and a PLP-mediated C–S lyase (EgtE) reaction. Our bioinformatic and biochemical analyses indicate that the fungus Neurospora crassa has a more concise ergothioneine biosynthetic pathway because its nonheme iron enzyme, Egt1, makes use of cysteine instead of γ-Glu-Cys as the substrate. Such a change of substrate preference eliminates the competition between ergothioneine and glutathione biosyntheses. In addition, we have identified the N. crassa C–S lyase (NCU11365) and reconstituted its activity in vitro, which makes the future ergothioneine production through metabolic engineering feasible.

Protein-reactive electrophiles are critical to chemical proteomic applications including activity-based protein profiling, site-selective protein modification, and covalent inhibitor development. Here, we explore the protein reactivity of a panel of aryl halides that function through a nucleophilic aromatic substitution (S(N)Ar) mechanism. We show that the reactivity of these electrophiles can be finely tuned by varying the substituents on the aryl ring. We identify p-chloro- and fluoronitrobenzenes and dichlorotriazines as covalent protein modifiers at low micromolar concentrations. Interestingly, investigating the site of labeling of these electrophiles within complex proteomes identified p-chloronitrobenzene as highly cysteine selective, whereas the dichlorotriazine favored reactivity with lysines. These studies illustrate the diverse reactivity and amino-acid selectivity of aryl halides and enable the future application of this class of electrophiles in chemical proteomics.

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MitoNEET is a small mitochondrial protein that has been identified recently as a target for the thiazolidinedione (TZD) class of diabetes drugs. MitoNEET also binds a unique 3His/1Cys-ligated [2Fe2S 2 ] cluster. Here we use protein film voltammetry (PFV) as a means to probe the redox properties of MitoNEET and demonstrate the direct impact of TZD drug binding upon the redox chemistry of the FeS cluster. Upon binding TZDs the midpoint potential at pH 7 is lowered by over 100 mV, shifting from ~ 0 mV to −100 mV. In contrast, a His87Cys mutant negates the ability of TZDs to affect the midpoint potential, suggesting a model of drug binding where His87 is critical to communication with the FeS center of MitoNEET.While the thiazolidinedione (TZD)1 drugs have been useful in the treatment of type 2 diabetes, their primary mode of action has been attributed to activation of peroxisome proliferator-activated receptor γ (PPARγ) (1-3). Recent appreciation of PPARγ-independent modes of TZD action (4) spurred the discovery of MitoNEET, a mitochondrial protein, through a cross-linking study with a photoactive form of the TZD drug pioglitazone ( Figure 1) (5). Intriguingly, MitoNEET bears a [2Fe-2S] cluster (6;7), and the cardiac mitochondria of mice lacking MitoNEET display dramatic decreases in respiratory function (6). The typical association of FeS proteins with redox events in biology, the growing appreciation of the interplay between mitochondrial dysfunction and type 2 diabetes (8) as well as the observation of oxidative stress in diabetic patients (9), led us to posit that MitoNEET may possess redox chemistry that is an important component of mitochondrial function, and that TZD drug binding may stabilize normal function of MitoNEET in diabetes. Here we describe the redox chemistry of the mitoNEET [2Fe-2S] cluster for the first time, demonstrate that TZD binding directly impacts the mitoNEET reduction potential, and show that TZD binding stabilizes the oxidized state of the wild-type protein.MitoNEET is a small ~17kD protein localized to the cytosolic face of the outer mitochondrial membrane by a single transmembrane helix (6). X-ray crystallographic analyses of the soluble portion of the protein ( Figure 1A) have demonstrated that mitoNEET