Adenosylcobalamin (AdoCbl) shows different photochemical behavior when free in solution compared to when it is bound to the photoregulatory protein CarH. In both cases, the initial photoproduct is cob(II)alamin and the 5'-deoxyadenosyl radical (Rios-Santacruz et al., Nature, 2026, 650, 1045-1052). However, in solution, the ultimate products are cob(II)alamin and the cyclic product, 5',8-cycloadenosine, while in the CarH protein, cob(I)alamin and the exocyclic methylene product 4',5'-anhydroadenosine are formed. This has led to speculation that alkene formation in CarH plays a protective role, preventing deleterious reactions of the 5'-deoxyadenosyl radical. Recently, we have found that the CarH-type reactivity (formation of alkene + cob(I)alamin) is not in fact unusual; we find this behavior across a range of alkylcobalamins (alkylCbls) (Mukherjee et al., Chem. Sci., 2026, 17, 5518-5531). In order to explore the factors driving these contrasting outcomes, we have characterized the photolytic reactivity of AdoCbl, both in a variety of solvents as well as in several AdoCbl-dependent proteins. In no case do we find evidence for CarH-type reactivity, even under conditions where other alkylCbls consistently generate cob(I)alamin + alkene. These findings indicate that the anomalous CarH behavior may be better understood not as CarH enforcing unusual reactivity on the cobalamin, but rather as CarH enabling the intrinsic alkyl-radical behavior that is otherwise quenched when the 5'-deoxyadenosyl radical is free in solution or bound in regular cobalamin-dependent proteins.

Inter-organellar communication is critical for cellular metabolism. One of the most abundant inter-organellar interactions occurs at the endoplasmic reticulum and mitochondria contact sites (ERMCSs). However, an understanding of the mechanisms governing ERMCS regulation and their roles in cellular metabolism is limited by a lack of tools that permit temporal induction and reversal. Through screening approaches, we identified fedratinib, an FDA-approved drug that dramatically increases ERMCS abundance by inhibiting the epigenetic modifier BRD4. Fedratinib rapidly and reversibly modulates mitochondrial and ER morphology, induces a distinct ER-mitochondria envelopment structure, and alters metabolic homeostasis. Moreover, ERMCS modulation depends on mitochondrial electron transport chain complex III function. Comparison of fedratinib activity to other reported inducers of ERMCSs revealed common mechanisms of induction and function, providing clarity to a growing body of experimental observations. In total, our results uncovered a novel epigenetic signaling pathway and an endogenous metabolic regulator that connects ERMCSs and cellular metabolism.

Colorectal cancer (CRC) cells are addicted to iron, which fuels nucleotide synthesis, mitochondrial respiration, and proliferation. Yet paradoxically, high intracellular iron is cytotoxic to most cells, raising the question of how CRC cells tolerate and exploit iron-rich environments. Ferroptosis, an iron-dependent form of cell death, is thought to mediate iron toxicity. However, whether most ferroptosis regulators, identified through synthetic chemical screens or small molecule activators, play a role in modulating iron toxicity, particularly in vivo, remains unclear. Here, using multi-omics profiling, CRISPR screening, and in vivo models, we uncover a heme-succinate dehydrogenase (SDH)-coenzyme Q (CoQ) axis that enables CRC cells to buffer iron-induced oxidative stress. Heme-dependent SDH reduces CoQ, which redistributes to mitochondrial and plasma membranes to detoxify lipid reactive oxygen species (ROS) as a radical-trapping antioxidant. These findings reveal that CRCs co-opt metabolic cofactors both for growth and for survival under physiologically toxic iron levels, uncovering new vulnerabilities for therapy.

Oxygen (O 2 ) sensing by hypoxia-inducible factor (HIF) is a principal mechanism by which aerobic organisms adjust cellular energy metabolism and adapt to O 2 limitation. In this study, we show that hydrogen sulfide (H 2 S), a product of host and microbial metabolism, profoundly influences the threshold for HIF-dependent hypoxia sensing by increasing intracellular O 2 . The dose-dependent destabilization of HIF by H 2 S is inversely correlated with sulfide quinone oxidoreductase, which oxidizes sulfide in the mitochondrion. Hypoxia sensors provide a semiquantitative estimate of the magnitude of H 2 S-induced perturbation. Thus, the O 2 concentration in cells grown in a 2% O 2 atmosphere is sensed as 5% or 15% O 2 in the presence of 25 or 100 ppm H 2 S, respectively. Sustained exposure to H 2 S elicits the hallmarks of hyperoxia-associated cytotoxicity, including loss of iron-sulfur proteins in cellular and murine models. H 2 S thus emerges as a powerful regulator of O 2 sensing and signaling with possible implications for dysregulation in O 2 toxicity diseases.

We asked a collection of redox biologists, “What do you think are the most exciting frontiers or the most needed developments in redox biology?” — here is what they said.

Teeming with microbes, the unique biogeography of the gut is shaped by interactions between diet, host and microbial metabolism. Hydrogen sulfide represents one such plane of interaction in the lower gut where it is largely the product of microbial activity. Sulfide oxidation by host epithelial cells helps shape a severely hypoxic luminal environment in which obligate anaerobes thrive and furnish among other products, butyrate, a fuel of choice for colonocytes. This metabolic symbiosis in healthy gut is supported by diet, and disrupted when the host sulfide oxidation capacity is exceeded, with resultant local and long-range impacts, including increased susceptibility to enteric pathogens and behavioral changes. Under homeostatic conditions, sulfide oxidation tunes host energy and redox metabolism that is corrupted under dysbiosis linked to gastrointestinal diseases. H2S could also be important for inducing a metabolic state change as in hibernating animals, by increasing energy storage in the form of reduced cofactors as well as increasing intracellular oxygen. In this review, we bracket luminal free sulfide exposure to colonocytes based on bioenergetic studies on colon-derived cells, discuss the microbial pathways for sulfide generation, and their interplay with dietary sulfur and host oxygen and redox metabolism.

The reactivity of copper, an essential micronutrient that undergoes facile cycling between Cu 1+ and Cu 2+ redox states, is carefully controlled within the confines of protein binding sites, and by sequestration in storage vesicles, or harnessed to kill pathogens by active pumping of Cu 1+ into phagosomes. We have discovered that hydrogen sulfide, a signaling metabolite generated in copious quantities at the host-microbiome interface, upregulates Cu accumulation in diffusely dispersed puncta across the cell, as visualized by X-ray fluorescence microscopy. The Cu is predominantly in the Cu 2+ state with oxygen/nitrogen ligands. Cu import occurs via the non- canonical ZNT1 transporter, while export, following sulfide withdrawal, is ATP7A-dependent. Cu accumulates at the apices of colon crypts in a mouse model of elevated sulfide exposure due to SQOR deficiency in the intestinal epithelium, establishing in vivo relevance. Our study reveals that sulfide is a dynamic regulator of the Cu pool, stimulating Cu 2+ influx into highly concentrated puncta.

Of the ~1,100 mitochondrial proteins, only a handful like PINK1 and ATFS-1 are known to stabilize and relocalize upon collapse of the proton motive force (PMF) to execute signaling roles. To systematically identify genes that increase exclusively at the protein level upon PMF collapse, we performed a joint proteomic and RNA-seq screen. The screen revealed 10 candidates (six mitochondrial), including two genes in vitamin B 12 metabolism - the B 12 chaperone MMADHC and cytosolic B 12 -dependent 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR). MMADHC is short-lived across cell types and we show that its levels increase with PMF collapse. MMADHC stabilization precedes PINK1 activation in a time course of increasing mtDNA depletion, suggesting greater sensitivity to PMF collapse. MMADHC accumulates in mitochondria with LONP1 inhibition but in the cytosol upon PMF collapse, likely due to mitochondrial import failure. Cytosol-stabilized MMADHC increases MTR levels and activity. Altogether, the mitochondrial PMF regulates the cytosolic B 12 -dependent MTR, integral to one-carbon metabolism, by controlling the stability and compartmentalization of the B 12 chaperone MMADHC.

Abstract MMUT is one of only two enzymes in the human proteome that depend on the B12 cofactor for activity. It catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA using 5′-deoxyadenosylcobalamin (AdoCbl) as a radical generator. Clinical mutations in MMUT are inherited in an autosomal recessive manner and are classified as B12-responsive mut– and B12 unresponsive mut0 subgroups. In this study, we report that missense mutations located at the same residue, i.e., Ala-137, are associated with distinct biochemical penalties. While the A137G mutation disables cofactor loading from the MMAB chaperone, it shows an ∼700-fold weaker affinity for binding AdoCbl from solution and exhibits an ∼700-fold lower activity than the wild-type enzyme. In contrast, the A137V variant binds AdoCbl tightly, can be partially loaded with cofactor from MMAB from solution, but exhibits ∼5000-fold lower activity, consistent with its mut0 classification. Despite the overall similarity in the crystal structure with wild-type MMUT, a close-up of the A137V active site reveals profound differences in the 5′-deoxyadenosyl moiety, which is held in a previously unseen pose. Structural analyses predict that while Gly-137 is too small to correctly position Tyr-110, critical for transition state stabilization, Val-137 is too large to even accommodate it, exemplifying the catalysis-permissive Goldilocks zone at this site. Our study predicts that the A137G variant might belong to the mut– subgroup and exhibit therapeutic responsiveness to a high dose of B12.

Tumor cells must occupy and thrive in a competitive microenvironment marked by limited metabolites, including essential amino acids like methionine. Using a leukemia suppression model and CRISPR screening, we found that the choline transporter SLC44A1 is overexpressed in leukemia patients and impacts leukemogenesis. Choline is an important nutrient for membrane synthesis and less commonly contributes to the methionine cycle. A metabolic analysis demonstrated that metabolites of the methionine pathway are significantly elevated in leukemic cells. Surprisingly, dietary restriction of methionine accelerated leukemogenesis in vivo. Choline can serve as an alternative source for methionine via the enzymatic activity of CHDH and BHMT. Under restrictive methionine conditions, BHMT and CHDH are significantly upregulated. In vivo, BHMT and CHDH are necessary for leukemia progression where they utilize choline as an alternative source to satisfy increased methionine demand. This pathway represents a vulnerability in cancer cells that may be exploited for therapeutic intervention.

Hydrogen sulfide (H2S) is a respiratory poison and also a product of our own metabolism. The toxicity of H2S is mitigated by the activity of mitochondrial sulfide quinone oxidoreductase (SQOR), which oxidizes H2S while concomitantly reducing coenzyme Q. An unusual cysteine trisulfide cofactor distinguishes SQOR from other members of the flavin disulfide reductase superfamily. In the opening step of the catalytic cycle, an estimated 105-fold rate enhancement is afforded by nucleophilic addition of the sulfide anion to the trisulfide versus a disulfide cofactor. The source of the bridging sulfane sulfur in the trisulfide and its mechanism of installation are, however, unknown. We report that H2S exposure (100 ppm corresponding to 20 μM dissolved H2S, 24 h) increases SQOR activity five- and twofold in human colon adenocarcinoma (HT-29) and transformed endothelial (EA.hy926) cells, respectively. Since activation is not accompanied by a corresponding increase in SQOR protein levels, we conclude that it involves a post-translational mechanism. CRISPR knockdowns of the sulfurtransferases (mercaptopyruvate sulfurtransferase and thiosulfate sulfurtransferase) rule out their involvement in SQOR activation. A combination of pharmacological inhibition and cystine supplementation studies points to the role of H2S rather than low molecular weight persulfides in regulating SQOR. We posit that the solvent accessibility and reactivity of the trisulfide make SQOR vulnerable to reversible inhibition. Our study supports a model for trisulfide installation and activation via cysteine oxidation and sulfide addition and reveals a heretofore unrecognized mechanism for autoregulating SQOR by H2S on demand.

Chaperones in trafficking pathways ensure specificity of transition metal loading and protection against adventitious side reactions. In B 12 , an essential cofactor for humans, a cobalt ion is coordinated to a >1.3 kDa tetrapyrrolic scaffold, posing logistical challenges for its translocation. In this study, we report the 3.4 Å crystal structure of the human MMACHC and MMADHC B 12 chaperones, tethered via a rarely seen covalent cobalt–sulfur bond. B 12 is bound in the base-off state to MMACHC, with Cys-261 on MMADHC serving as the upper axial ligand; the lower-axial position is vacant. The propensity of thiolato-cobalamin derivatives bound to MMACHC to undergo spontaneous decomposition via general acid catalysis or reduction/oxidation chemistry is averted in the interprotein complex with MMADHC. An exposed face in the complex suggests an exit route for B 12 . No known clinical variants localize to the interprotein interface, consistent with the cobalt–sulfur bond being key to forming the high-affinity complex.