Absence Of Cofactors Research Articles

Re-assessing viologens for modern bio-electrocatalysis.

Viologens, 1,1'-disubstituted-4,4'-bipyridinium salts, are organic redox species that can be used in place of NADPH as mediators for redox enzymes. In this study, using the reduction of oxidized glutathione by glutathione reductase as a model system, a rationally designed library of viologens covering a range of polarities and functional groups were explored as electron transfer mediators for bio-electrocatalysis. Through a series of electrochemical investigations, the reduction potential was found to be the primary determining factor for electron transfer between the viologen and enzyme. Through enhancing the solubility of viologen such that the fully reduced state remained soluble, we demonstrate a much-widened window of useable viologen potentials. In doing so, we describe for the first time a highly efficient electron transfer to a flavoenzyme promoting the catalytic reaction in the absence of co-factors. As such, our study provides a platform for broadening the scope for using viologens as mediating agents for electrochemically-driven enzymatic processes.

Wheat-dependent exercise-induced anaphylaxis: subtypes, diagnosis, and management.

Wheat-dependent exercise-induced anaphylaxis (WDEIA) is an IgE-mediated food allergy with allergic symptoms ranging from intermittent urticaria to severe anaphylaxis that occurs when wheat ingestion is combined with augmenting cofactors such as exercise, non-steroidal anti-inflammatory drugs, or alcohol. In most cases, patients are identified by sensitization to ω5-gliadins in the gluten fraction of wheat. ω5-gliadin-negative subtypes of WDEIA are often difficult to diagnose and may be caused by Tri a 14 (wheat lipid transfer protein), after percutaneous sensitization with hydrolyzed wheat proteins, or, in rare cases, by cross-reactivity to grass pollen. Diagnosis is established based on the patients' history in combination with serum IgE profile, skin testing, basophil activation tests, and challenge tests with cofactors. Individual dietary counselling remains the central pillar in the management of WDEIA patients. A completely wheat-free diet is a possible option. However, this appears to promote tolerance less than continued regular consumption of gluten-containing cereals in the absence of cofactors. All patients should have an emergency set for self-treatment including an adrenaline autoinjector and receive adequate instruction. More data are needed on sublingual immunotherapy for WDEIA, a potentially promising therapeutic prospect. This article provides an overview of current knowledge on the diagnosis and management of WDEIA including an optimized challenge protocol using wheat gluten and cofactors.

In silico drug screen reveals potential competitive MTHFR inhibitors for clinical repurposing

MTHFR (Methylenetetrahydrofolate reductase) is a pivotal enzyme involved in one-carbon metabolism, which is critical for the proliferation of cancer cells. In line with this, published literature showed that MTHFR knockdown caused impaired growth of multiple types of cancer cells. Moreover, higher MTHFR expression levels were linked to shorter overall survival in hepatocellular carcinoma, adrenocortical carcinoma, and low-grade glioma, bringing the need to design MTHFR inhibitors as a possible treatment option. No competitive inhibitors of MTHFR have been reported as of today. This study aimed to identify potential competitive MTHFR inhibitor candidates using an in silico drug screen. A total of 30470 molecules containing biogenic compounds, FDA-approved drugs, and those in clinical trials were screened against the catalytic pocket of MTHFR in the presence and absence of cofactors. Binding energy and ADMET analysis revealed that Vilanterol (β2-adrenergic agonist), Selexipag (prostacyclin receptor agonist), and Ramipril Diketopiperazine (ACE inhibitor) are potential competitive inhibitors of MTHFR. Molecular dynamics analyses and MM-PBSA calculations with these compounds particularly revealed the amino acids between 285-290 for ligand binding and highlighted Vilanterol as the strongest candidate for MTHFR inhibition. Our results could guide the development of novel MTHFR inhibitor compounds, which could be inspired by the drugs brought into the spotlight here. More importantly, these potential candidates could be quhickly tested as a repurposing strategy in pre-clinical and clinical studies of the cancers mentioned above. Communicated by Ramaswamy H. Sarma

Cofactors, age at onset, allergic comorbidities and gender are different in patients sensitized to omega-5 gliadin and Pru p 3

Aim of this study is to clarify the impact of cofactors on allergic reactions in patients sensitized to LTP and ω-5-gliadin. We retrospectively examined the charts of our outpatients from January 2015 to July 2019 and identified 157 patients seen for urticaria/angioedema or anaphylaxis after a meal, in presence or absence of cofactors and sensitized to LTPs (Pru p 3 and/or Tri a 14) and/or ω-5-gliadin (Tri a 19). we compared LTPs-positive patients and those sensitized to Tri a 19 in order to assess the difference in cofactors involved and in frequency of clinical presentation. Our data show that exercise is the most frequent cofactor in FDEIAn and the involvement of exercise, alcohol and multiple cofactors was more frequently found in males than in females. We found that exercise, pollen counts peaks and multiple cofactors were more often related to anaphylaxis than mild reactions. Finally, we performed a comparison between patients LTPs+ and Tri a 19+ that showed in the latter group a lower frequency of allergic comorbidities, a higher median age at the onset of symptoms and frequency of alcohol exposure. Our data show that the search for possible cofactors involved in food allergy is essential not only for diagnostic purposes, but also for risk assessment strategies.

Structures of lactaldehyde reductase, FucO, link enzyme activity to hydrogen bond networks and conformational dynamics.

A group-III iron containing 1,2-propanediol oxidoreductase, FucO, (also known as lactaldehyde reductase) from Escherichia coli was examined regarding its structure-dynamics-function relationships in the catalysis of the NADH-dependent reduction of (2S)-lactaldehyde. Crystal structures of FucO variants in the presence or absence of cofactors have been determined, illustrating large domain movements between the apo and holo enzyme structures. Different structures of FucO variants co-crystallized with NAD+ or NADH together with substrate further suggest dynamic properties of the nicotinamide moiety of the coenzyme that are important for the reaction mechanism. Modelling of the native substrate (2S)-lactaldehyde into the active site can explain the stereoselectivity exhibited by the enzyme, with a critical hydrogen bond interaction between the (2S)-hydroxyl and the side-chain of N151, as well as the previously experimentally demonstrated pro-(R) selectivity in hydride transfer from NADH to the aldehydic carbon. Furthermore, the deuterium kinetic isotope effect of hydride transfer suggests that reduction chemistry is the main rate-limiting step for turnover which is not the case in FucO catalysed alcohol oxidation. We further propose that a water molecule in the active site - hydrogen bonded to a conserved histidine (H267) and the 2'-hydroxyl of the coenzyme ribose - functions as a catalytic proton donor in the protonation of the product alcohol. A hydrogen bond network of water molecules and the side-chains of amino acid residues D360 and H267 links bulk solvent to this proposed catalytic water molecule.

Reconstruction of dynamic regulatory networks reveals signaling-induced topology changes associated with germ layer specification.

SummaryElucidating regulatory relationships between transcription factors (TFs) and target genes is fundamental to understanding how cells control their identity and behavior. Unfortunately, existing computational gene regulatory network (GRN) reconstruction methods are imprecise, computationally burdensome, and fail to reveal dynamic regulatory topologies. Here, we present Epoch, a reconstruction tool that uses single-cell transcriptomics to accurately infer dynamic networks. We apply Epoch to identify the dynamic networks underpinning directed differentiation of mouse embryonic stem cells (ESCs) guided by multiple signaling pathways, and we demonstrate that modulating these pathways drives topological changes that bias cell fate potential. We also find that Peg3 rewires the pluripotency network to favor mesoderm specification. By integrating signaling pathways with GRNs, we trace how Wnt activation and PI3K suppression govern mesoderm and endoderm specification, respectively. Finally, we identify regulatory circuits of patterning and axis formation that distinguish in vitro and in vivo mesoderm specification.

Mechanistic Insight into Metal Ion-Catalyzed Transamination.

Several classes of biological reactions that are mediated by an enzyme and a co-factor can occur, to a slower extent, not only without the enzyme but even without the co-factor, under catalysis by metal ions. This observation has led to the proposal that metabolic pathways progressively evolved from using inorganic catalysts to using organocatalysts of increasing complexity. Transamination, the biological process by which ammonia is transferred between amino acids and α-keto acids, has a mechanism that has been well studied under enzyme/co-factor catalysis and under co-factor catalysis, but the metal ion-catalyzed variant was generally studied mostly at high temperatures (70-100°C), and the details of its mechanism remained unclear. Here, we investigate which metal ions catalyze transamination under conditions relevant to biology (pH 7, 20-50 °C) and study the mechanism in detail. Cu2+, Ni2+, Co2+, and V5+ were identified as the most active metal ions under these constraints. Kinetic, stereochemical, and computational studies illuminate the mechanism of the reaction. Cu2+ and Co2+ are found to predominantly speed up the reaction by stabilizing a key imine intermediate. V5+ is found to accelerate the reaction by increasing the acidity of the bound imine. Ni2+ is found to do both to a limited extent. These results show that direct metal ion-catalyzed amino group transfer is highly favored even in the absence of co-factors or protein catalysts under biologically compatible reaction conditions.

Co-factor-free aggregation of tau into seeding-competent RNA-sequestering amyloid fibrils

Pathological aggregation of the protein tau into insoluble aggregates is a hallmark of neurodegenerative diseases. The emergence of disease-specific tau aggregate structures termed tau strains, however, remains elusive. Here we show that full-length tau protein can be aggregated in the absence of co-factors into seeding-competent amyloid fibrils that sequester RNA. Using a combination of solid-state NMR spectroscopy and biochemical experiments we demonstrate that the co-factor-free amyloid fibrils of tau have a rigid core that is similar in size and location to the rigid core of tau fibrils purified from the brain of patients with corticobasal degeneration. In addition, we demonstrate that the N-terminal 30 residues of tau are immobilized during fibril formation, in agreement with the presence of an N-terminal epitope that is specifically detected by antibodies in pathological tau. Experiments in vitro and in biosensor cells further established that co-factor-free tau fibrils efficiently seed tau aggregation, while binding studies with different RNAs show that the co-factor-free tau fibrils strongly sequester RNA. Taken together the study provides a critical advance to reveal the molecular factors that guide aggregation towards disease-specific tau strains.

Metabolism of HER2‐Selective Kinase Inhibitor Tucatinib in Human Liver Fractions

As breast cancer continues to take the lives of women all over the world, targeted therapies have become the standard of care for many metastatic and end stage cancers. Tucatinib is a recently FDA-approved tyrosine kinase inhibitor, which targets the HER2 receptor and is used in combination to treat advanced or metastatic HER2-positive breast cancer. Preliminary studies indicate that tucatinib is metabolized primarily by cytochrome P450 (CYP) 2C8, with minor contributions from CYP3A and aldehyde oxidase (AO). The goal of this study was to identify the metabolites formed from tucatinib in vitro, and to determine the enzymatic contributions to tucatinib metabolism. To characterize CYP-mediated and AO-mediated metabolites of tucatinib, in vitro reaction phenotyping assays were performed. Tucatinib was incubated with human liver microsomes (HLM), human liver S9 fractions (HLS9), and recombinant CYP enzymes over time in the presence and absence of cofactor, NADPH. Incubations were also conducted in HLS9 in the presence and absence of the non-selective CYP inhibitor 1-aminobenzotriazole (ABT) to estimate the overall CYP contribution. Reaction products were analyzed using LC/MS-MS. Two tucatinib metabolites, M1 and M2, were detected from initial reaction phenotyping experiments; M1 was the primary metabolite generated by recombinant CYP2C8, and M2 was formed in part by CYP3A4. These metabolites were further separated by LC-MS/MS into distinct peaks: M1a, M1b, M2a, and M2b. M1a and M2b were generated in an NADPH-dependent manner, and formation was reduced by ABT. M2a appeared to be formed through a non-CYP, NADPH-independent route. Interestingly, M2a formation was higher in the presence of ABT compared to control incubations without the CYP inhibitor. Collectively, these data suggest that M1a and M2b are CYP-mediated metabolites of tucatinib, while M2a may have partial contribution from AO. Due to an impurity in the tucatinib standard, M1b could not be fully characterized. Further experiments are needed to confirm the metabolic pathways of tucatinib and identify the source of M1b. Future investigations using individual donor tissues will provide insight into the interindividual variability in metabolism of tucatinib, which has relevance for HER2-positive breast cancer treatment.

PBX3 in Cancer.

PBX3 is a homeodomain-containing transcription factor of the pre-B cell leukemia (PBX) family, members of which have extensive roles in early development and some adult processes. A number of features distinguish PBX3 from other PBX proteins, including the ability to form specific and stable interactions with DNA in the absence of cofactors. PBX3 has frequently been reported as having a role in the development and maintenance of a malignant phenotype, and high levels of PBX3 tumor expression have been linked to shorter overall survival in cancer. In this review we consider the similarities and differences in the function of PBX3 in different cancer types and draw together the core signaling pathways involved to help provide a better insight into its potential as a therapeutic target.

Short bZIP homologue of sulfur regulator Met4 from Ogataea parapolymorpha does not depend on DNA-binding cofactors for activating genes in sulfur starvation.

The acquisition of sulfur from environment and its assimilation is essential for fungal growth and activities. Here, we describe novel features of the regulatory network of sulfur metabolism in Ogataea parapolymorpha, a thermotolerant methylotrophic yeast with high resistance to harsh environmental conditions. A short bZIP protein (OpMet4p) of O. parapolymorpha, displaying the combined structural characteristics of yeast and filamentous fungal Met4 homologues, plays a key role as a master regulator of cell homeostasis during sulfur limitation, but also its function is required for the tolerance of various stresses. Domain swapping analysis, combined with deletion analysis of the regulatory domains and genes encoding OpCbf1p, OpMet28p, and OpMet32p, indicated that OpMet4p does not require the interaction with these DNA-binding cofactors to induce the expression of sulfur genes, unlike the Saccharomyces cerevisiae Met4p. ChIP analysis confirmed the notion that OpMet4p, which contains a canonical bZIP domain, can bind the target DNA in the absence of cofactors, similar to homologues in other filamentous fungi. Collectively, the identified unique features of the O. parapolymorpha regulatory network, as the first report on the sulfur regulation by a short yeast Met4 homologue, provide insights into conservation and divergence of the sulfur regulatory networks among diverse ascomycetous fungi.

Calcium exerts a strong influence upon phosphohydrolase gene abundance and phylogenetic diversity in soil

The mechanisms by which microbial communities maintain functions within the context of changing environments are key to a wide variety of environmental processes. In soil, these mechanisms support fertility. Genes associated with hydrolysis of organic phosphoesters represent an interesting set of genes with which to study maintenance of function in microbiomes. Here, we shown that the richness of ecotypes for each gene varies considerably in response to application of manure and various inorganic fertilizer combinations. We show, at unprecedented phylogenetic resolution, that phylogenetic diversity of phosphohydrolase genes are more responsive to soil management and edaphic factors than the taxonomic biomarker 16S rRNA gene. Available phosphorus – assessed by measuring Olsen-P - exerted some influence on alkaline phosphatase distribution: however, consistent and significant differences were observed in gene abundance between treatments that were inconsistent with bioavailable orthophosphate being the dominant factor determining gene abundance. Instead, we observed gene niche separation which was most strongly associated with soil exchangeable calcium. Our study suggests that the bioavailability of enzyme cofactors (exchangeable calcium in the case of phoD, phoX and βPPhy studied here) influence the abundance of genes in soil microbial communities; in the absence of cofactors, genes coding for alternative enzyme families that do not require the limiting cofactor (for example, non-specific acid phosphatases which require vanadate) become more abundant.

Structural and biochemical characterization of Rv0187, an O-methyltransferase from Mycobacterium tuberculosis

Catechol O-methyltransferase (COMT) is widely distributed in nature and installs a methyl group onto one of the vicinal hydroxyl groups of a catechol derivative. Enzymes belonging to this family require two cofactors for methyl transfer: S-adenosyl-l-methionine as a methyl donor and a divalent metal cation for regiospecific binding and activation of a substrate. We have determined two high-resolution crystal structures of Rv0187, one of three COMT paralogs from Mycobacterium tuberculosis, in the presence and absence of cofactors. The cofactor-bound structure clearly locates strontium ions and S-adenosyl-l-homocysteine in the active site, and together with the complementary structure of the ligand-free form, it suggests conformational dynamics induced by the binding of cofactors. Examination of in vitro activities revealed promiscuous substrate specificity and relaxed regioselectivity against various catechol-like compounds. Unexpectedly, mutation of the proposed catalytic lysine residue did not abolish activity but altered the overall landscape of regiospecific methylation.

Molecular dynamics characterization of the SAMHD1 Aicardi–Goutières Arg145Gln mutant: structural determinants for the impaired tetramerization

Aicardi-Goutières syndrome, a rare genetic disorder characterized by calcification of basal ganglia, results in psychomotor delays and epilepsy states from the early months of children life. This disease is caused by mutations in seven different genes encoding proteins implicated in the metabolism of nucleic acids, including SAMHD1. Twenty SAMHD1 gene variants have been discovered and in this work, a structural characterization of the SAMHD1 Aicardi-Goutières Arg145Gln mutant is reported by classical molecular dynamics simulation. Four simulations have been carried out and compared. Two concerning the wild-type SAMHD1 form in presence and absence of cofactors, in order to explain the role of cofactors in the SAMHD1 assembly/disassembly process and, two concerning the Arg145Gln mutant, also in presence and absence of cofactors, in order to have an accurate comparison with the corresponding native forms. Results show the importance of native residue Arg145 in maintaining the tetramer, interacting with GTP cofactor inside allosteric sites. Replacement of arginine in glutamine gives rise to a loosening of GTP-protein interactions, when cofactors are present in allosteric sites, whilst in absence of cofactors, the occurrence of intra and inter-chain interactions is observed in the mutant, not seen in the native enzyme, making energetically unfavourable the tetramerization process.

Role of the CBP catalytic core in intramolecular SUMOylation and control of histone H3 acetylation

The histone acetyl transferases CREB-binding protein (CBP) and its paralog p300 play a critical role in numerous cellular processes. Dysregulation of their catalytic activity is associated with several human diseases. Previous work has elucidated the regulatory mechanisms of p300 acetyltransferase activity, but it is not known whether CBP activity is controlled similarly. Here, we present the crystal structure of the CBP catalytic core encompassing the bromodomain (BRD), CH2 (comprising PHD and RING), HAT, and ZZ domains at 2.4-Å resolution. The BRD, PHD, and HAT domains form an integral structural unit to which the RING and ZZ domains are flexibly attached. The structure of the apo-CBP HAT domain is similar to that of acyl-CoA-bound p300 HAT complexes and shows that the acetyl-CoA binding site is stably formed in the absence of cofactor. The BRD, PHD, and ZZ domains interact with small ubiquitin-like modifier 1 (SUMO-1) and Ubc9, and function as an intramolecular E3 ligase for SUMOylation of the cell cycle regulatory domain 1 (CRD1) of CBP, which is located adjacent to the BRD. In vitro HAT assays suggest that the RING domain, the autoregulatory loop (AL) within the HAT domain, and the ZZ domain do not directly influence catalytic activity, whereas the BRD is essential for histone H3 acetylation in nucleosomal substrates. Several lysine residues in the intrinsically disordered AL are autoacetylated by the HAT domain. Upon autoacetylation, acetyl-K1596 (Ac-K1596) binds intramolecularly to the BRD, competing with histones for binding to the BRD and acting as a negative regulator that inhibits histone H3 acetylation.

A new mode of DNA binding distinguishes Capicua from other HMG-box factors and explains its mutation patterns in cancer.

HMG-box proteins, including Sox/SRY (Sox) and TCF/LEF1 (TCF) family members, bind DNA via their HMG-box. This binding, however, is relatively weak and both Sox and TCF factors employ distinct mechanisms for enhancing their affinity and specificity for DNA. Here we report that Capicua (CIC), an HMG-box transcriptional repressor involved in Ras/MAPK signaling and cancer progression, employs an additional distinct mode of DNA binding that enables selective recognition of its targets. We find that, contrary to previous assumptions, the HMG-box of CIC does not bind DNA alone but instead requires a distant motif (referred to as C1) present at the C-terminus of all CIC proteins. The HMG-box and C1 domains are both necessary for binding specific TGAATGAA-like sites, do not function via dimerization, and are active in the absence of cofactors, suggesting that they form a bipartite structure for sequence-specific binding to DNA. We demonstrate that this binding mechanism operates throughout Drosophila development and in human cells, ensuring specific regulation of multiple CIC targets. It thus appears that HMG-box proteins generally depend on auxiliary DNA binding mechanisms for regulating their appropriate genomic targets, but that each sub-family has evolved unique strategies for this purpose. Finally, the key role of C1 in DNA binding also explains the fact that this domain is a hotspot for inactivating mutations in oligodendroglioma and other tumors, while being preserved in oncogenic CIC-DUX4 fusion chimeras associated to Ewing-like sarcomas.

The production of S-equol from daidzein is associated with a cluster of three genes in Eggerthella sp. YY7918.

Daidzein (DZN) is converted to equol (EQL) by intestinal bacteria. We previously reported that Eggerthella sp. YY7918, which is found in human feces, is an EQL-producing bacterium and analyzed its whole genomic sequence. We found three coding sequences (CDSs) in this bacterium that showed 99% similarity to the EQL-producing enzymes of Lactococcus sp. 20-92. These identified CDSs were designated eqlA, eqlB, and eqlC and thought to encode daidzein reductase (DZNR), dihydrodaidzein reductase (DHDR), and tetrahydrodaidzein reductase (THDR), respectively. These genes were cloned into pColdII. Recombinant plasmids were then introduced into Escherichia coli BL21 (DE3) and DZNR, DHDR, and THDR were expressed and purified by 6×His-Tag chromatography. We confirmed that these three enzymes were involved in the conversion of DZN to EQL. Purified DZNR converted DZN to dihydrodaizein (DHD) in the presence of NADPH. DHDR converted DHD to tetrahydrodaizein (THD) in the presence of NADPH. Neither enzyme showed activities with NADH. THDR converted THD in the absence of cofactors, NAD(P)H, and also produced DHD as a by-product. Thus, we propose that THDR is not a reductase but a new type of dismutase. The GC content of these clusters was 64%, similar to the overall genomic GC content for Eggerthella and Coriobacteriaceae (56–60%), and higher than that for Lactococcus garvieae (39%), even though the gene cluster showed 99% similarity to that in Lactococcus sp. 20-92. Taken together, our results indicate that the gene cluster associated with EQL production evolved in high-GC bacteria including Coriobacteriaceae and was then laterally transferred to Lactococcus sp. 20-92.

Intrinsic unfoldase/foldase activity of the chaperonin GroEL directly demonstrated using multinuclear relaxation-based NMR

The prototypical chaperonin GroEL assists protein folding through an ATP-dependent encapsulation mechanism. The details of how GroEL folds proteins remain elusive, particularly because encapsulation is not an absolute requirement for successful re/folding. Here we make use of a metastable model protein substrate, comprising a triple mutant of Fyn SH3, to directly demonstrate, by simultaneous analysis of three complementary NMR-based relaxation experiments (lifetime line broadening, dark state exchange saturation transfer, and Carr-Purcell-Meinboom-Gill relaxation dispersion), that apo GroEL accelerates the overall interconversion rate between the native state and a well-defined folding intermediate by about 20-fold, under conditions where the "invisible" GroEL-bound states have occupancies below 1%. This is largely achieved through a 500-fold acceleration in the folded-to-intermediate transition of the protein substrate. Catalysis is modulated by a kinetic deuterium isotope effect that reduces the overall interconversion rate between the GroEL-bound species by about 3-fold, indicative of a significant hydrophobic contribution. The location of the GroEL binding site on the folding intermediate, mapped from (15)N, (1)HN, and (13)Cmethyl relaxation dispersion experiments, is composed of a prominent, surface-exposed hydrophobic patch.

Functional insights from high resolution structures of mouse protein arginine methyltransferase 6.

PRMT6 is a protein arginine methyltransferase involved in transcriptional regulation, human immunodeficiency virus pathogenesis, DNA base excision repair, and cell cycle progression. Like other PRMTs, PRMT6 is overexpressed in several cancer types and is therefore considered as a potential anti-cancer drug target. In the present study, we described six crystal structures of PRMT6 from Mus musculus, solved and refined at 1.34 Å for the highest resolution structure. The crystal structures revealed that the folding of the helix αX is required to stabilize a productive active site before methylation of the bound peptide can occur. In the absence of cofactor, metal cations can be found in the catalytic pocket at the expected position of the guanidinium moiety of the target arginine substrate. Using mass spectrometry under native conditions, we show that PRMT6 dimer binds two cofactor and a single H4 peptide molecules. Finally, we characterized a new site of in vitro automethylation of mouse PRMT6 at position 7.

Experimental Support for a Single Electron-Transfer Oxidation Mechanism in Firefly Bioluminescence.

Firefly luciferase produces light by converting substrate beetle luciferin into the corresponding adenylate that it subsequently oxidizes to oxyluciferin, the emitter of bioluminescence. We have confirmed the generally held notions that the oxidation step is initiated by formation of a carbanion intermediate and that a hydroperoxide (anion) is involved. Additionally, structural evidence is presented that accounts for the delivery of oxygen to the substrate reaction site. Herein, we report key convincing spectroscopic evidence of the participation of superoxide anion in a related chemical model reaction that supports a single electron-transfer pathway for the critical oxidative process. This mechanism may be a common feature of bioluminescence processes in which light is produced by an enzyme in the absence of cofactors.