The Human Knockout Gene CLYBL Connects Itaconate to Vitamin B12.

CLYBL encodes a ubiquitously expressed mitochondrial enzyme, conserved across all vertebrates, whose cellular activity and pathway assignment are unknown. Its homozygous loss is tolerated in seemingly healthy individuals, with reduced circulating B12 levels being the only and consistent phenotype reported to date. Here, by combining enzymology, structural biology, and activity-based metabolomics, we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells. Cells lacking CLYBL accumulate citramalyl-CoA, an intermediate in the C5-dicarboxylate metabolic pathway that includes itaconate, a recently identified human anti-microbial metabolite and immunomodulator. We report that CLYBL loss leads to a cell-autonomous defect in the mitochondrial B12 metabolism and that itaconyl-CoA is a cofactor-inactivating, substrate-analog inhibitor of the mitochondrial B12-dependent methylmalonyl-CoA mutase (MUT). Our work de-orphans the function of human CLYBL and reveals that a consequence of exposure to the immunomodulatory metabolite itaconate is B12 inactivation.

Promoting a MOSAIC future in science.

Discoveries at the frontiers of science and finding solutions to pressing biomedical problems will be accelerated when talent, which is widely distributed, is better aligned with opportunities. Strategies to enhance a MOSAIC (Maximizing Opportunities for Scientific and Academic Independent Careers) professoriate and diversify the biomedical landscape are discussed.

H2S preconditioning induces long-lived perturbations in O2 metabolism.

Hydrogen sulfide exposure in moderate doses can induce profound but reversible hypometabolism in mammals. At a cellular level, H2S inhibits the electron transport chain (ETC), augments aerobic glycolysis, and glutamine-dependent carbon utilization via reductive carboxylation; however, the durability of these changes is unknown. We report that despite its volatility, H2S preconditioning increases P50(O2), the O2 pressure for half-maximal cellular respiration, and has pleiotropic effects on oxidative metabolism that persist up to 24 to 48 h later. Notably, cyanide, another complex IV inhibitor, does not induce this type of metabolic memory. Sulfide-mediated prolonged fractional inhibition of complex IV by H2S is modulated by sulfide quinone oxidoreductase, which commits sulfide to oxidative catabolism. Since induced hypometabolism can be beneficial in disease settings that involve insufficient or interrupted blood flow, our study has important implications for attenuating reperfusion-induced ischemic injury and/or prolonging the shelf life of biologics like platelets.

Gas regulation of complex II reversal via electron shunting to fumarate in the mammalian ETC.

The electron transport chain (ETC) is a major currency converter that exchanges the chemical energy of fuel oxidation to proton motive force and, subsequently, ATP generation, using O2 as a terminal electron acceptor. Discussed herein, two new studies reveal that the mammalian ETC is forked. Hypoxia or H2S exposure promotes the use of fumarate as an alternate terminal electron acceptor. The fumarate/succinate and CoQH2/CoQ redox couples are nearly iso-potential, revealing that complex II is poised for facile reverse electron transfer, which is sensitive to CoQH2 and fumarate concentrations. The gas regulators, H2S and •NO, modulate O2 affinity and/or inhibit the electron transfer rate at complex IV. Their induction under hypoxia suggests a mechanism for how traffic at the ETC fork can be regulated.

Sulfide oxidation promotes hypoxic angiogenesis and neovascularization.

Angiogenic programming in the vascular endothelium is a tightly regulated process for maintaining tissue homeostasis and is activated in tissue injury and the tumor microenvironment. The metabolic basis of how gas signaling molecules regulate angiogenesis is elusive. Here, we report that hypoxic upregulation of ·NO in endothelial cells reprograms the transsulfuration pathway to increase biogenesis of hydrogen sulfide (H2S), a proangiogenic metabolite. However, decreased H2S oxidation due to sulfide quinone oxidoreductase (SQOR) deficiency synergizes with hypoxia, inducing a reductive shift and limiting endothelial proliferation that is attenuated by dissipation of the mitochondrial NADH pool. Tumor xenografts in whole-body (WBCreSqorfl/fl) and endothelial-specific (VE-cadherinCre-ERT2Sqorfl/fl) Sqor-knockout mice exhibit lower mass and angiogenesis than control mice. WBCreSqorfl/fl mice also exhibit decreased muscle angiogenesis following femoral artery ligation compared to control mice. Collectively, our data reveal the molecular intersections between H2S, O2 and ·NO metabolism and identify SQOR inhibition as a metabolic vulnerability for endothelial cell proliferation and neovascularization.

Bivalent molecular mimicry by ADP protects metal redox state and promotes coenzyme B12 repair.

Control over transition metal redox state is essential for metalloprotein function and can be achieved via coordination chemistry and/or sequestration from bulk solvent. Human methylmalonyl-Coenzyme A (CoA) mutase (MCM) catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA using 5'-deoxyadenosylcobalamin (AdoCbl) as a metallocofactor. During catalysis, the occasional escape of the 5'-deoxyadenosine (dAdo) moiety leaves the cob(II)alamin intermediate stranded and prone to hyperoxidation to hydroxocobalamin, which is recalcitrant to repair. In this study, we have identified the use of bivalent molecular mimicry by ADP, coopting the 5'-deoxyadenosine and diphosphate moieties in the cofactor and substrate, respectively, to protect against cob(II)alamin overoxidation on MCM. Crystallographic and electron paramagnetic resonance (EPR) data reveal that ADP exerts control over the metal oxidation state by inducing a conformational change that seals off solvent access, rather than by switching five-coordinate cob(II)alamin to the more air stable four-coordinate state. Subsequent binding of methylmalonyl-CoA (or CoA) promotes cob(II)alamin off-loading from MCM to adenosyltransferase for repair. This study identifies an unconventional strategy for controlling metal redox state by an abundant metabolite to plug active site access, which is key to preserving and recycling a rare, but essential, metal cofactor.

Acidity of persulfides and its modulation by the protein environments in sulfide quinone oxidoreductase and thiosulfate sulfurtransferase.

Persulfides (RSSH/RSS-) participate in sulfur metabolism and are proposed to transduce hydrogen sulfide (H2S) signaling. Their biochemical properties are poorly understood. Herein, we studied the acidity and nucleophilicity of several low molecular weight persulfides using the alkylating agent, monobromobimane. The different persulfides presented similar pKa values (4.6-6.3) and pH-independent rate constants (3.2-9.0 × 103 M-1 s-1), indicating that the substituents in persulfides affect properties to a lesser extent than in thiols because of the larger distance to the outer sulfur. The persulfides had higher reactivity with monobromobimane than analogous thiols and putative thiols with the same pKa, providing evidence for the alpha effect (enhanced nucleophilicity by the presence of a contiguous atom with high electron density). Additionally, we investigated two enzymes from the human mitochondrial H2S oxidation pathway that form catalytic persulfide intermediates, sulfide quinone oxidoreductase and thiosulfate sulfurtransferase (TST, rhodanese). The pH dependence of the activities of both enzymes was measured using sulfite and/or cyanide as sulfur acceptors. The TST half-reactions were also studied by stopped-flow fluorescence spectroscopy. Both persulfidated enzymes relied on protonated groups for reaction with the acceptors. Persulfidated sulfide quinone oxidoreductase appeared to have a pKa of 7.8 ± 0.2. Persulfidated TST presented a pKa of 9.38 ± 0.04, probably due to a critical active site residue rather than the persulfide itself. The TST thiol reacted in the anionic state with thiosulfate, with an apparent pKa of 6.5 ± 0.1. Overall, our study contributes to a fundamental understanding of persulfide properties and their modulation by protein environments.

Rapid HPLC method reveals dynamic shifts in coenzyme Q redox state.

Ubiquinol or coenzyme Q (CoQ) is a lipid-soluble electron carrier in the respiratory chain and an electron acceptor for various enzymes in metabolic pathways that intersect at this cofactor hub in the mitochondrial inner membrane. The reduced form of CoQ is an antioxidant, which protects against lipid peroxidation. In this study, we have optimized a UV-detected HPLC method for CoQ analysis from biological materials, which involves a rapid single-step extraction into n-propanol followed by direct sample injection onto a column. Using this method, we have measured the oxidized, reduced, and total CoQ pools and monitored shifts in the CoQ redox status in response to cell culture conditions and bioenergetic perturbations. We find that hypoxia or sulfide exposure induces a reductive shift in the intracellular CoQ pool. The effect of hypoxia is, however, rapidly reversed by exposure to ambient air. Interventions at different loci in the electron transport chain can induce sizeable redox shifts in the oxidative or reductive direction, depending on whether they are up- or downstream of complex III. We have also used this method to confirm that CoQ levels are higher and more reduced in murine heart versus brain. In summary, the availability of a convenient HPLC-based method described herein will facilitate studies on CoQ redox dynamics in response to environmental, nutritional, and endogenous alterations.

Disease-causing cystathionine ß-synthase linker mutations impair allosteric regulation.

Cystathionine ß-synthase (CBS) catalyzes the committing step in the transsulfuration pathway, which is important for clearing homocysteine and furnishing cysteine. The transsulfuration pathway also generates H2S, a signaling molecule. CBS is a modular protein with a heme and pyridoxal phosphate-binding catalytic core, which is separated by a linker region from the C-terminal regulatory domain that binds S-adenosylmethionine (AdoMet), an allosteric activator. Recent cryo-EM structures reveal that CBS exists in a fibrillar form and undergoes a dramatic architectural rearrangement between the basal and AdoMet-bound states. CBS is the single most common locus of mutations associated with homocystinuria, and, in this study, we have characterized three clinical variants (K384E/N and M391I), which reside in the linker region. The native fibrillar form is destabilized in the variants, and differences in their limited proteolytic fingerprints also reveal conformational alterations. The crystal structure of the truncated K384N variant, lacking the regulatory domain, reveals that the overall fold of the catalytic core is unperturbed. M391I CBS exhibits a modest (1.4-fold) decrease while the K384E/N variants exhibit a significant (∼8-fold) decrease in basal activity, which is either unresponsive to or inhibited by AdoMet. Pre-steady state kinetic analyses reveal that the K384E/N substitutions exhibit pleiotropic effects and that the differences between them are expressed in the second half reaction, that is, homocysteine binding and reaction with the aminoacrylate intermediate. Together, these studies point to an important role for the linker in stabilizing the higher-order oligomeric structure of CBS and enabling AdoMet-dependent regulation.

Regulation of the redox metabolome and thiol proteome by hydrogen sulfide.

Overproduction of reactive oxygen species and compromised antioxidant defenses perturb intracellular redox homeostasis and is associated with a myriad of human diseases as well as with the natural process of aging. Hydrogen sulfide (H2S), which is biosynthesized by organisms ranging from bacteria to man, influences a broad range of physiological functions. A highly touted molecular mechanism by which H2S exerts its cellular effects is via post-translational modification of the thiol redox proteome, converting cysteine thiols to persulfides, in a process referred to as protein persulfidation. The physiological relevance of this modification in the context of specific signal transmission pathways remains to be rigorously established, while a general protective role for protein persulfidation against hyper-oxidation of the cysteine proteome is better supported. A second mechanism by which H2S modulates redox homeostasis is via remodeling the redox metabolome, targeting the electron transfer chain and perturbing the major redox nodes i.e. CoQ/CoQH2, NAD+/NADH and FAD/FADH2. The metabolic changes that result from H2S-induced redox changes fan out from the mitochondrion to other compartments. In this review, we discuss recent developments in elucidating the roles of H2S and its oxidation products on redox homeostasis and its role in protecting the thiol proteome.

A redox cycle with complex II prioritizes sulfide quinone oxidoreductase-dependent H2S oxidation.

The dual roles of H2S as an endogenously synthesized respiratory substrate and as a toxin raise questions as to how it is cleared when the electron transport chain is inhibited. Sulfide quinone oxidoreductase (SQOR) catalyzes the first step in the mitochondrial H2S oxidation pathway, using CoQ as an electron acceptor, and connects to the electron transport chain at the level of complex III. We have discovered that at high H2S concentrations, which are known to inhibit complex IV, a new redox cycle is established between SQOR and complex II, operating in reverse. Under these conditions, the purine nucleotide cycle and the malate aspartate shuttle furnish fumarate, which supports complex II reversal and leads to succinate accumulation. Complex II knockdown in colonocytes decreases the efficiency of H2S clearance while targeted knockout of complex II in intestinal epithelial cells significantly decreases the levels of thiosulfate, a biomarker of H2S oxidation, to approximately one-third of the values seen in serum and urine samples from control mice. These data establish the physiological relevance of this newly discovered redox circuitry between SQOR and complex II for prioritizing H2S oxidation and reveal the quantitatively significant contribution of intestinal epithelial cells to systemic H2S metabolism.

Cobalt-Sulfur Coordination Chemistry Drives B12 Loading onto Methionine Synthase.

Cobalt-sulfur (Co-S) coordination is labile to both oxidation and reduction chemistry and is rarely seen in nature. Cobalamin (or vitamin B12) is an essential cobalt-containing organometallic cofactor in mammals and is escorted via an intricate network of chaperones to a single cytoplasmic target, methionine synthase. In this study, we report that the human cobalamin trafficking protein, MMADHC, exploits the chemical lability of Co-S coordination for cofactor off-loading onto methionine synthase. Cys-261 on MMADHC serves as the ß-axial ligand to cobalamin. Complex formation between MMADHC and methionine synthase is signaled by loss of the lower axial nitrogen ligand, leading to five-coordinate thiolato-cobalamin. Nucleophilic displacement by the vicinal thiolate, Cys-262, completes cofactor transfer to methionine synthase and release of a cysteine disulfide-containing MMADHC. The physiological relevance of this mechanism is supported by clinical variants of MMADHC, which impair cofactor binding and off-loading, explaining the molecular basis of the associated homocystinuria.

A growth chamber for chronic exposure of mammalian cells to H2S.

H2S is a redox-active signaling molecule that exerts an array of cellular and physiological effects. While intracellular H2S concentrations are estimated to be in the low nanomolar range, intestinal luminal concentrations can be significantly higher due to microbial metabolism. Studies assessing H2S effects are typically conducted with a bolus treatment with sulfide salts or slow releasing sulfide donors, which are limited by the volatility of H2S, and by potential off-target effects of the donor molecules. To address these limitations, we describe the design and performance of a mammalian cell culture incubator for sustained exposure to 20-500 ppm H2S (corresponding to a dissolved sulfide concentrations of ∼4-120 µM in the cell culture medium). We report that colorectal adenocarcinoma HT29 cells tolerate prolonged exposure to H2S with no effect on cell viability after 24 h although ≥50 ppm H2S (∼10 µM) restricts cell proliferation. Even the lowest concentration of H2S used in this study (i.e. ∼4 µM) significantly enhanced glucose consumption and lactate production, revealing a much lower threshold for impacting cellular energy metabolism and activating aerobic glycolysis than has been previously appreciated from studies with bolus H2S treatment regimens.

Coordination Chemistry Controls Coenzyme B12 Synthesis by Human Adenosine Triphosphate:Cob(I)alamin Adenosyltransferase.

Cobalamin (or vitamin B12)-dependent enzymes and trafficking chaperones exploit redox-linked coordination chemistry to control the cofactor reactivity during catalysis and translocation. As the cobalt oxidation state decreases from 3+ to 1+, the preferred cobalamin geometry changes from six- to four-coordinate (4-c). In this study, we reveal the sizable thermodynamic gain that accrues for human adenosine triphosphate (ATP):cob(I)alamin adenosyltransferase (or MMAB) by enforcing an unfavorable 4-c cob(II)alamin geometry. MMAB-bound cob(II)alamin is reduced to the supernucleophilic cob(I)alamin intermediate during the synthesis of 5'-deoxyadenosylcobalamin. Herein, we report the first experimentally determined reduction potential for 4-c cob(II)alamin (-325 ± 9 mV), which is 180 mV more positive than for the five-coordinate (5-c) water-liganded species. The redox potential of MMAB-bound cob(II)alamin is within the range of adrenodoxin, which we demonstrate functions as an electron donor. We also show that stabilization of 5-c cob(II)alamin by a subset of MMAB patient variants compromises the reduction by adrenodoxin, explaining the underlying pathogenic mechanism.

Mobile loop dynamics in adenosyltransferase control binding and reactivity of coenzyme B12.

Cobalamin is a complex organometallic cofactor that is processed and targeted via a network of chaperones to its dependent enzymes. AdoCbl (5'-deoxyadenosylcobalamin) is synthesized from cob(II)alamin in a reductive adenosylation reaction catalyzed by adenosyltransferase (ATR), which also serves as an escort, delivering AdoCbl to methylmalonyl-CoA mutase (MCM). The mechanism by which ATR signals that its cofactor cargo is ready (AdoCbl) or not [cob(II)alamin] for transfer to MCM, is not known. In this study, we have obtained crystallographic snapshots that reveal ligand-induced ordering of the N terminus of Mycobacterium tuberculosis ATR, which organizes a dynamic cobalamin binding site and exerts exquisite control over coordination geometry, reactivity, and solvent accessibility. Cob(II)alamin binds with its dimethylbenzimidazole tail splayed into a side pocket and its corrin ring buried. The cosubstrate, ATP, enforces a four-coordinate cob(II)alamin geometry, facilitating the unfavorable reduction to cob(I)alamin. The binding mode for AdoCbl is notably different from that of cob(II)alamin, with the dimethylbenzimidazole tail tucked under the corrin ring, displacing the N terminus of ATR, which is disordered. In this solvent-exposed conformation, AdoCbl undergoes facile transfer to MCM. The importance of the tail in cofactor handover from ATR to MCM is revealed by the failure of 5'-deoxyadenosylcobinamide, lacking the tail, to transfer. In the absence of MCM, ATR induces a sacrificial cobalt-carbon bond homolysis reaction in an unusual reversal of the heterolytic chemistry that was deployed to make the same bond. The data support an important role for the dimethylbenzimidazole tail in moving the cobalamin cofactor between active sites.

Patient mutations in human ATP:cob(I)alamin adenosyltransferase differentially affect its catalytic versus chaperone functions.

Human ATP:cob(I)alamin adenosyltransferase (ATR) is a mitochondrial enzyme that catalyzes an adenosyl transfer to cob(I)alamin, synthesizing 5'-deoxyadenosylcobalamin (AdoCbl) or coenzyme B12. ATR is also a chaperone that escorts AdoCbl, transferring it to methylmalonyl-CoA mutase, which is important in propionate metabolism. Mutations in ATR lead to methylmalonic aciduria type B, an inborn error of B12 metabolism. Our previous studies have furnished insights into how ATR protein dynamics influence redox-linked cobalt coordination chemistry, controlling its catalytic versus chaperone functions. In this study, we have characterized three patient mutations at two conserved active site residues in human ATR, R190C/H, and E193K and obtained crystal structures of R190C and E193K variants, which display only subtle structural changes. All three mutations were found to weaken affinities for the cob(II)alamin substrate and the AdoCbl product and increase KM(ATP). 31P NMR studies show that binding of the triphosphate product, formed during the adenosylation reaction, is also weakened. However, although the kcat of this reaction is significantly diminished for the R190C/H mutants, it is comparable with the WT enzyme for the E193K variant, revealing the catalytic importance of Arg-190. Furthermore, although the E193K mutation selectively impairs the chaperone function by promoting product release into solution, its catalytic function might be unaffected at physiological ATP concentrations. In contrast, the R190C/H mutations affect both the catalytic and chaperoning activities of ATR. Because the E193K mutation spares the catalytic activity of ATR, our data suggest that the patients carrying this mutation are more likely to be responsive to cobalamin therapy.

The mitochondrial NADH pool is involved in hydrogen sulfide signaling and stimulation of aerobic glycolysis.

Hydrogen sulfide is synthesized by enzymes involved in sulfur metabolism and oxidized via a dedicated mitochondrial pathway that intersects with the electron transport chain at the level of complex III. Studies with H2S are challenging since it is volatile and also reacts with oxidized thiols in the culture medium, forming sulfane sulfur species. The half-life of exogenously added H2S to cultured cells is unknown. In this study, we first examined the half-life of exogenously added H2S to human colonic epithelial cells. In plate cultures, H2S disappeared with a t1/2 of 3 to 4 min at 37 °C with a small fraction being trapped as sulfane sulfur species. In suspension cultures, the rate of abiotic loss of H2S was slower, and we demonstrated that sulfide stimulated aerobic glycolysis, which was sensitive to the mitochondrial but not the cytoplasmic NADH pool. Oxidation of mitochondrial NADH using the genetically encoded mito-LbNOX tool blunted the cellular sensitivity to sulfide-stimulated aerobic glycolysis and enhanced its oxidation to thiosulfate. In contrast, sulfide did not affect flux through the oxidative pentose phosphate pathway or the TCA cycle. Knockdown of sulfide quinone oxidoreductase, which commits H2S to oxidation, sensitized cells to sulfide-stimulated aerobic glycolysis. Finally, we observed that sulfide decreased ATP levels in cells. The dual potential of H2S to activate oxidative phosphorylation at low concentrations, but inhibit it at high concentrations, suggests that it might play a role in tuning electron flux and, therefore, cellular energy metabolism, particularly during cell proliferation.

Protein-aggregating ability of different protoporphyrin-IX nanostructures is dependent on their oxidation and protein-binding capacity.

Porphyrias are rare blood disorders caused by genetic defects in the heme biosynthetic pathway and are associated with the accumulation of high levels of porphyrins that become cytotoxic. Porphyrins, due to their amphipathic nature, spontaneously associate into different nanostructures, but very little is known about the cytotoxic effects of these porphyrin nanostructures. Previously, we demonstrated the unique ability of fluorescent biological porphyrins, including protoporphyrin-IX (PP-IX), to cause organelle-selective protein aggregation, which we posited to be a major mechanism by which fluorescent porphyrins exerts their cytotoxic effect. Herein, we tested the hypothesis that PP-IX-mediated protein aggregation is modulated by different PP-IX nanostructures via a mechanism that depends on their oxidizing potential and protein-binding ability. UV-visible spectrophotometry showed pH-mediated reversible transformations of PP-IX nanostructures. Biochemical analysis showed that PP-IX nanostructure size modulated PP-IX-induced protein oxidation and protein aggregation. Furthermore, albumin, the most abundant serum protein, preferentially binds PP-IX dimers and enhances their oxidizing ability. PP-IX binding quenched albumin intrinsic fluorescence and oxidized His-91 residue to Asn/Asp, likely via a previously described photo-oxidation mechanism for other proteins. Extracellular albumin protected from intracellular porphyrinogenic stress and protein aggregation by acting as a PP-IX sponge. This work highlights the importance of PP-IX nanostructures in the context of porphyrias and offers insights into potential novel therapeutic approaches.

Hydrogen sulfide stimulates lipid biogenesis from glutamine that is dependent on the mitochondrial NAD(P)H pool.

Mammalian cells synthesize H2S from sulfur-containing amino acids and are also exposed to exogenous sources of this signaling molecule, notably from gut microbes. As an inhibitor of complex IV in the electron transport chain, H2S can have a profound impact on metabolism, suggesting the hypothesis that metabolic reprogramming is a primary mechanism by which H2S signals. In this study, we report that H2S increases lipogenesis in many cell types, using carbon derived from glutamine rather than from glucose. H2S-stimulated lipid synthesis is sensitive to the mitochondrial NAD(P)H pools and is enabled by reductive carboxylation of α-ketoglutarate. Lipidomics analysis revealed that H2S elicits time-dependent changes across several lipid classes, e.g., upregulating triglycerides while downregulating phosphatidylcholine. Direct analysis of triglyceride concentration revealed that H2S induces a net increase in the size of this lipid pool. These results provide a mechanistic framework for understanding the effects of H2S on increasing lipid droplets in adipocytes and population studies that have pointed to a positive correlation between cysteine (a substrate for H2S synthesis) and fat mass.