Nitric Oxide Inhibition of Glycyl Radical Enzymes and Their Activases

Innate immune response cells produce high concentrations of the free radical nitric oxide (NO) in response to pathogen infection. The antimicrobial properties of NO include non-specific damage to essential biomolecules and specific inactivation of enzymes central to aerobic metabolism. However, the molecular targets of NO in a naerobic metabolism are less understood. Here, we demonstrate that the Escherichia coli glycyl radical enzyme pyruvate formate lyase (PFL) , which catalyzes the anaerobic metabolism of pyruvate, is irreversibly inhibited by NO. Using electron paramagnetic resonance and site-directed mutagenesis we show that NO destroys the glycyl radical of PFL. The activation of PFL by its cognate radical S- adenosyl-L-methionine-dependent activating enzyme (PFL-AE) is also inhibited by NO, resulting in the conversion of the es- sential iron-sulfur cluster to dinitrosyl iron complexes. Whole-cell EPR and metabolic flux analyses of anaerobically growing Escherichia coli show that PFL and PFL -AE are inhibited by physiologically relevant levels of NO in bacterial cell cultures, resulting in diminished growth and a metabolic shift to lactate fermentation . The class III ribonucleoti de reductase (RNR) glycyl radical enzyme and its corresponding RNR-AE are also inhibited by NO in a mechanism analogous to those observed in PFL and PFL- AE, which likely contributes to the bacteriostatic effect of NO. Based on the similarities in reactivity of the PFL/RNR and PFL-AE/RNR-AE enzymes with NO, the mechanism of inactivation by NO appears to be general to the respective enzyme classes. The results implicate an immunological role of NO in inhibiting glycyl radical enzyme chemistry in the gut.

Opportunistic pathogens infecting the gastrointestinal and respiratory tracks, such as Shigella dysenteriae, Salmo- nella enterica, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, are facultative anaerobes.1–5 This metabolic flexibility allows for aerobic pathogen transmis- sion and subsequent establishment and proliferation in the anaerobic or microaerobic environment of the host mucosal epithelium-microbiome interface.6,7 Dysregulation of the microbiome at this interface induce s a host innate immune response, resulting in the release of reactive oxygen species (ROS, e.g. H 2O2)8,9 and reactive nitrogen species ( RNS, e.g. NO),10–12 both of which have pleiotropic and hormetic, yet still undefined roles in pathophysiology. 13,14 For microor- ganisms adapted to anaerobic metabolism, ROS disrupt re- dox homeostasis and damage biomolecules and essential cofactors. 15 The effect(s) of RNS, such as NO, on anaerobic metabolism are less defined and may have practical impli- cations in therapeutic developments to manage or treat dis- eases. 15–18 During anaerobic glycolysis, many bacteria and archaea in the human gut, at least partially, metabolize pyruvate to acetyl-coenzyme A (acetyl -CoA) and formate through the action of pyruvate formate lyase (PFL).19–21 PFL is a member of the glycyl radical enzyme (GRE) family, requiring a post- translationally installed glycyl radical for activity .22–24 This essential radical is installed by a specific activating enzyme, PFL-AE, a member of the radical S- adenosyl-L-methionine (SAM)-dependent enzyme superfamily. 25–27 The acetyl-CoA product can be used for substrate level phosphorylation to form adenosine triphosphate (ATP) and serve as a n elec- tron sink to recycle nicotinamide adenine dinucleotide (NAD+), or provide carbon in anabolic biosynthesis. 28 The formate product can be expelled as a waste product, either as formate or carbon dioxide and hydrogen, or used as an electron and carbon source. 29–31 Anaerobic growth in organ- isms that utilize PFL is often possible in the absence of PFL, provided a suitable electron acceptor is present, such as ni- trate, or acetate is available for acetyl-CoA biosynthesis. During infection, PFL is up- regulated in many opportun- istic pathogens. The deletion of pflA (PFL-AE) and pflB (PFL) genes results in a loss of virulence in some pathogens , fur- ther suggesting PFL provides a virulent fitness ad- vantage. 32–36 Given its role in central metabolism and path- ogenesis, and the inherent reactivity of amino acid radicals, we hypothesize that PFL is a molecular target of the im- mune response. The glycyl radical of PFL is extremely sen- sitive to O 2, cleaving the enzyme polypeptide chain at the glycyl α-carbon, but to our knowledge, no other ROS have been investigated.37,38 The radical SAM PFL -AE contains an essential [Fe 4S4] cofactor and is also sensitive to inactiva- tion by O2, suggesting that both PFL and PFL-AE are suscep- tible to inactivation by O2, and potentially other ROS. 19 Evi- dence for the reactivity of the RNS NO with PFL or PFL-AE, .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639758doi: bioRxiv preprint however, is indirect. In a study of S. aureus, exposure to NO under anaerobic conditions results in the diversion of me- tabolism from ethanol fermentation via PFL to lactate pro- duction, and in a murine infection model, S. aureus was non- pathogenic in the absence of an NO -inducible lactate dehy- drogenase.39 These results suggest that NO inhibits PFL in vivo, with implications in pathogenic disease progression. Here, we report the reactivity of NO with active glycyl radical-containing PFL (aPFL) from E. coli, PFL-AE, and ad- ditional representative members of the GRE and radical SAM enzyme families. In vitro, the addition of an NO donor to aPFL results in the complete loss of the glycyl radical and enzyme activity. The reaction appears to be diffusion -lim- ited and specific to the PFL glycyl radical. We observe simi- lar reactivity with the E. coli class III ribonucleotide reduc- tase (RNR), suggesting the mechanism of irreversible inhi- bition is common to the GRE family. We also demonstrate that PFL-AE and RNR- AE are irreversibly inhibited by NO, although through a slower process, involving dinitrosyl iron products, known inhibition products of [Fe 4S4] cluster-con- taining enzymes associated with aerobic metabolism such as aconitase and succinate dehydrogenase . 40–42 The inhibi- tion of PFL and PFL-AE was also observed in vivo by whole- cell electron paramagnetic resonance (EPR) spectroscopy, revealing the quenching of the glycyl radical signal and the concomitant formation of dinitrosyl iron complexes (DNICs) in PFL and PFL- AE overexpressing cells. This inhi- bition was accompanied by a shift in the metabolic products of anaerobically growing E. coli from acetate, formate , and ethanol to lactate production. These findings suggest that NO inhibits anaerobic microbial metabolism through multi- ple mechanisms, contributing to the antimicrobial activity of NO in the host immune response against pathogenic in- fections in anaerobic or microaerobic environments. EXPERIMENTAL Materials. Electrocompetent DH5α and BL21(DE3) E. coli and NEBuilder® HiFi DNA Assembly Master Mix were pur- chased from New England Biolabs. Carbenicillin and L-arab- inose were purchased from GoldBio. Chloramphenicol, kan- amycin and agarose were purchased from Apex Bioresearch Products. LB Miller broth, citrate synthase ( porcine heart), malate dehydrogenase (porcine heart), bovine serum albu- min, myoglobin (equine heart) , alcohol dehydrogenase (Saccharomyces cerevisiae ), tris(hydroxymethyl)amino- methane base (Tris), β -nicotinamide adenine dinucleotide (NAD+), S -(5′-adenosyl)-L-methionine (SAM) iodide salt, KH2PO4, Triton X -100, glycerol, MgCl 2·6H2O, oxamic acid, sodium pyruvate, sodium formate, sodium lactate, ethanol, malic acid, iodoacetamide, LC-MS grade trifluoroacetic acid (TFA), dithiothreitol (DTT), L-cysteine, (NH4)FeII(SO4)2, for- mic acid , 4-hydroxy-TEMPO, urea , sodium dithionite (NaDT), sodium borohydride (NaBH 4), glycine, glucose, MgSO4, CaCl2, biotin, thiamin, Na2HPO4, NH4Cl, ethylenedia- minetetraacetic acid (EDTA) , FeCl 3·6H2O, ZnCl 2, CuCl2·2H2O, CoCl 2·6H2O, H 3BO3, MnCl2·6H2O, iron IC P standards (TraceCERT) , 70% trace metal -free nitric acid, isopropyl-β-D-1-thiogalactopyranoside (IPTG), cytidine tri- phosphate (CTP), adenosine triphosphate (ATP), cytidine, 2′-deoxycytidine (dC), and Amicon Ultra centrifugal filter units were purchased from Millipore Sigma. 5-Deazaribofla- vin was obtained from Santa Cruz Biotechnologies . Sequencing grade modified trypsin was purchased from Promega Corporation. Coenzyme A was purchased from Co- Ala Biosciences. HiTrap desalting 5 mL columns were pur- chased from Cytiva Life Sciences. Nickel nitrilotriacetic acid (Ni-NTA) agarose resin was purchased from Qiagen . His- Pur™ Cobalt Resin and 0.5 mL Zeba™ spin desalting columns of 7 kDa molecular weight cut-off (MWCO) were purchased from Thermo Scientific. Adenosine (A) was purchased from VWR. Diethylammonium (Z) -1-(N,N-diethylamino)diazen- 1-ium-1,2-diolate (NONOate) was purchased from Cayman Chemicals. SapphireAmp® Fast PCR master mix was pur- chased from Takara Bio. Vivaspin 20 filtration units were purchased from Sartorius. The acetic acid assay kit (ACS Manual format) was purchased from Megazyme. Calf alka- line phosphatase was purchased from Roche. HPLC -grade water with 0.1% TFA and acetonitrile with 0.1% TFA were purchased from Honeywell. Milli-Q water ( >17 MΩ) was used for preparing all solutions. The plasmid pCAL-n-EK en- coding the pflA gene was a gift from Dr . Joan Broderick. 43 The pflB gene (Uniprot ID P09373) and the mutants C 418S and C419S were cloned into the plasmid pCM8 previously .44 The plasmid pCm2 NikJ was available from a previous study.45 The Pseudomonas sp. 101 formate dehydrogenase (FDH) gene (Uniprot ID P33160) was codon optimized and synthesized by Integrated DNA Technologies. The plasmids pFGET19_Ulp1 and pHYRSF53 were a gift from Hideo Iwai (Addgene plasmid # 64697 and # 64696, respectively ). 46 The TEVSH plasmid was a gift from Dr. Helena Berglund (Addgene plasmid # 125194). 47 The plasmid pDB1282 was a gift from Dr. Squire Booker and previously constructed in the laboratory of Dr. Dennis Dean.48,49 The plasmid pET28a- EcNrdD and pN9 -EcNrdG were a gift from Dr. JoAnne Stubbe50. The plasmid pLZ113 harboring the D176G-I177L- F178W LDH w as a gift from Dr. Han Li. 51 Construction of P la smids. To produce and purify PFL - AE, RNR-AE and FDH, we followed similar cloning strate- gies. Each gene was cloned into the pHYRSF53 plasmid us- ing Gibson assembly.52 These plasmids yield expressed pro- teins with an N-terminal 6× polyhistidine tag, up-stream of a small ubiquitin -like modifying (SUMO) protein fusion, termed SUMO -PFL-AE, SUMO -RNR-AE and SUMO -FDH re- spectively. The genes w ere cloned into the plasmid replac- ing the gene of the protein of interest encoded in pHYRSF53, downstream of the SUMO gene and in the same open read- ing frame by Gibson assembly. The DNA fragment contain- ing the genes (fragments) and the plasmid backbone (vec- tor) were cloned using the primers listed in the Supporting Information Table S1. The PCR-amplified complementary DNA fragments were assembled using NEBuilder® HiFi DNA Assembly Master Mix following the manufacturer ’s instructions. Assembl ed reaction products were transformed into E. coli DH5α cells and streaked o nto LB-agar plates supplemented with 50 μg/mL kanamycin. Successful transformants were identi- fied using colony PCR with the SapphireAmp fast PCR -hot- start master mix, as directed by the manufacturer, and the fidelity of the cloning was confirmed by Sanger sequencing through UC Berkeley DNA Sequencing Facility using the T7 promoter/terminator primers. Protein Expression and Purification. The expression and purification of the TEV protease and the ubiquitin-like .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639758doi: bioRxiv preprint protease 1 ( ULP1) were performed as previously de- scribed.46,47 The protein expression and purification of PFL wild type (wt, 77 ± 3 μmol/min×mg normalized to G●) and the serine (S) mutants C 418S and C419S, as well as PFL -AE, were carried out as previously described without further modifications.44 The enzyme NikJ (2.6 ± 0.1 Fe/protein) was expressed and purified as reported before using the pCm2 plasmid.45 Lactate dehydrogenase (LDH, 1,330 ± 30 s‒1) was expressed and purified as described before. 53 Expression and purification of FDH (2.3 μmol/min×mg), RNR (1,350 ± 90 nmol/min ×mg normalized to G ●), RNR -AE (2.2 ± 0.1 Fe/protein) and PFL-AE (2.1 ± 0.2 Fe/protein) are detailed in Supporting Information Supplemental Methods. Pure proteins concentration in water was determined using the Edelhoch method54 but using the extinction coefficients for tryptophan (W) and tyrosine (Y) determined by Pace.55 Iron Quantification. We quantified iron by inductively coupled plasma optic al e mission spectroscopy (ICP -OES). Typically, 8 nmol of protein was digested by adding 86 μL of 70% (v/v) trace metal-free nitric acid and incubated over- night at room temperature followed by 2 h incubation at 90 °C. Once samples returned to room temperature , 70 μL of 30% (v/v) hydrogen peroxide w as added and incubated at 90 °C for 1 h. Finally, water was added to a final weight of 3 g and analyzed in an Agilent 5800 ICP -OES in the following configuration: read time 5 s, RF power 1.45 k W, stabiliza- tion time 15 s, in axial viewing mode, nebulizer flow 0.7 L/min, plasma flow 12 L/min, and auxiliary flow 1 L/min. A calibration curve using iron standards in 2% nitric acid, with varying concentrations between 12.5 ppb and 1 ppm was used to quantitate iron in protein samples. PFL Activation . All experiments with glycyl radical en- zymes or activator enzymes were performed in a VAC At- mospheres glovebox (< 2 ppm O 2), unless otherwise de- scribed. We activated PFL photochemically by mixing 25 μM PFL, 2.5 μM PFL-AE, 2 mM SAM, 20 mM oxamate , and 100 μM 5-deazariboflavin in activation buffer composed of 100 mM Tris, 100 mM KCl, 10 mM DTT, and 8% (w/v) glycerol at pH 7.6 in a 50 mL beaker, volumes ranging from 800 μL to 2 mL were activated each time, producing a layer of pro- tein sample of 0.6 mm to 1.4 mm. The activation mixture was then exposed to a 1 W 405 nm LED light (Thor Labs) located approximately 5 cm above the protein sample for 1.5 h at room temperature. The lamp irradiance is 760 mW over the sample surface area . The extent of PFL activation was estimated by activity assays and EPR quantitation of the resultant G● (routinely 0.8-1.0 G●/PFL homodimer). We measured PFL activity spectrophotometrically using a multi-enzyme assay that couples the PFL- dependent for- mation of a cetyl-CoA from pyruvate to the production of NADH by the oxidation of malate to oxaloacetate and con- densation of oxaloacetate and acetyl-CoA to citrate and CoA by malate dehydrogenase and citrate synthase, respec- tively, as previously reported . 56–58 The 400 μL assay solu- tion contained 10 mM DTT, 1 mM NAD +, 10 mM malate, 2 U/mL citrate synthase, 30 U/mL malate dehydrogenase and 0.05 mg/mL bovine serum albumin in 100 mM Tris buffer, adjusted to pH 8.1. Reactions were initiated by adding 3 nM aPFL (based on G ●) and the rate of NADH production was calculated based on the UV absorption at 340 nm using an NADH extinction coefficient of 6.2 mM –1 cm–1 via a custom fiber-coupled Ocean Optics QEPro spectrophotometer and a DH-2000-BAL light source.59 Inactivation of PFL by NO. For enzyme inactivation ki- netics we buffer-exchanged aPFL into 100 mM Tris and 100 mM KCl at pH 7.6 using a Zeba Spin d esalting column to a final concentration of 10 μM aPFL and maintained the solu- tion at 30 °C using a water bath. The NONOate was then added to the aPFL solution to an estimated final concentra- tion of either 100 μM or 1 mM from a stock solution in 100 mM glycine bu ffer pH 10 .0. Aliquots of the reaction were sampled from 10 s to 26 min after mixing and immediately diluted 50-fold in buffer consisting of 100 mM glycine at pH 10.0 supplemented with 0.2 mg/mL of reduced deoxymyo- globin (deoxyMb) to stop the release of NO and bind any free NO. We then determined the remaining aPFL activity as described above. To estimate the NO concentration during the decomposi- tion of the NONOate we used deoxyMb as an NO indicator by measuring the conversion of the deoxyMb h eme Soret peak shift from 431 nm to 421 nm upon binding NO. 60 First, a solution of 10 mg/mL of reduced myoglobin was prepared by mixing oxidized myoglobin, quantitated by the heme So- ret absorbance at 409 nm, 61 with 10 mM NaDT in buffer con- sisting of 100 mM Tris and 100 mM KCl at pH 7.6 and then desalted to the same buffer without NaDT. The resulting de- oxyMb concentration was redetermined by the heme Soret absorbance at 431 nm. 62 To measure NO release from NON- Oate, 20 μM of deoxyMb was incubated with 100 μM or 1 mM NONOate in the same way as for aPFL. Aliquots were sampled during the reaction from 10 s to 26 min and imme- diately diluted 50 -fold in buffer consisting of 100 mM gly- cine at pH 10.0 to stop the release of NO. NO concentration was estimated using the NO-Mb Soret absorbance of the heme-NO adduct at 421 nm. 63 The loss of the aPFL G ● upon reaction with NO was ana- lyzed by UV -vis absorption, monitoring the characteristic G● absorption feature at 360 nm. 64 In this assay, a solution of 55 μM aPFL in 100 mM T ris and 100 mM KCl pH 7.6 was prepared in a quartz cuvette and NONOate was added to a final concentration of 500 μM at room temperature. The re- action was followed over time by using a custom fiber-cou- pled Ocean Optics QEPro spectrophotometer and a DH- 2000-BAL light source. Inactivated samples were also analyzed by EPR. S imilar inactivation assays were prepared using 20 μM of aPFL wt, C 418S or C419S and 200 μM DEA NONOate. Samples (250 μL) were then transferred to EPR tubes and flash frozen in liq- uid N2-cooled isopentane (< ‒130 °C) at different reaction times and analyzed as described below. X-Band EPR Spectroscopy . All EPR samples were pre- pared in a VAC Atmosphere glovebox with < 2 ppm of O2 in 4 mm o.d. quartz EPR tubes and frozen in liquid N 2-cooled isopentane (< ‒130 °C) . EPR spectra of the samples were collected using a Bruker EMXplus EPR spectrometer at 100 K with a microwave frequency between 9.38-9.44 GHz , power of 20 μW or 2 mW, modulation amplitude of 2 G, modulation frequency of 100 kHz, time constant of 0.01 ms, scan time of 20 s, and conversion time of 16 ms. All reported spectra are the average of 30 scans . Spin quantitation was computed from the double integral of the first harmonic sig- nal and referenced to a 4-hydroxy-TEMPO standard. All EPR .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639758doi: bioRxiv preprint spectral simulations were performed using EasySpin 6.0.0 software, and confidence intervals and standard deviations of the fitting parameters are reported. 65 Liquid Chromatography-Tandem Mass Spectrometry. We searched for covalent modifications of the glycine radi- cal motif tryptic peptide in reactions of aPFL treated with NO by using trypsin digestion followed by liquid chroma- tography and electrospray ionization quadrupole time -of- flight tandem mass spectrometry (LC -MS/MS) on a Shi- madzu LCMS-9030 q-TOF system . Samples of 25 μM aPFL were treated with 100 μM or 1 mM NONOate and incubated at room temperature for 10 min to 1 h. Reactions were stopped by mixing 5 μL of sample with 20 μL of 8 M urea in 100 mM ammonium bicarbonate and incubated for 1 h at 25 °C. Alternatively , some samples were treated with 10 mM TCEP, 10 mM DTT, or 10 mM sodium borohydride to reduce cysteines or possible nitroxyl adducts. For samples in re- ducing conditions cysteines were alkylated with 15 mM io- doacetamide for 60 min in the dark, and the reaction was quenched by adding 10 mM DTT or TCEP and incubated for 10 min. The samples were diluted to < 2 M urea using 100 mM ammonium bicarbonate and 0.16 μg of trypsin was added and the protein was digested at 37 °C overnight. Re- actions were stopped by adding formic acid to a final con- centration of 1% (v/v). The digested samples (2 -5 μg at 0.4- 1 μg/μL) wer e in- jected into the LC-MS/MS. The UPLC stationary phase was a Shim-pack Arata C18 column (2.2 μm, 150 mm × 2.0 mm). The peptides were resolved by a linear gradient composed of a solution of H 2O with 0.1% ( v/v) formic acid ( mobile phase A) and a solution of acetonitrile with 0.1% (v/v) for- mic acid (mobile phase B), from 1-30% B over 233 min, with a flow rate of 0.2 mL/min at 60 °C. Eluent from the LC was injected directly into the q -ToF. The mass spectrometer in- terface settings were as follow: nebulizing gas flow 2 L/min, heating gas flow 10 L/min, interface temperature 100 – 300 °C, and a desolvation temperature of 526 °C. The interface voltage was set to 4.5 V and the DL temperature to 250 °C. The spectrometer was run in data -dependent acquisition mode (DDA) with positive polarity using an event time of 0.35 s (MS 1). Five DDA events between 200 and 1500 m/z , with a threshold of 200 counts and ions with charges be- tween 1 and 6, were selected for fragmentation (MS 2) using a Q1 transmission window of 1 m/z. The MS2 collision en- ergy was set to 35 ± 17 V and MS 2 ions were detected be- tween 200 and 1,500 m/z. The LC -MS/MS data was analyzed using the Shimadzu Lab solutions Postrun software. The MS1 and MS2 ions with different expected modifications on the glycyl radical tryp- tic peptide were calculated using Skyline. 66,67 The DDA- selected MS1 ions were fragmented and the ions matching predicted m/z for glycyl radical tryptic peptides were man- ually compared to the calculated MS 2 spectra from Skyline. Inactivation of PFL-AE by NO. We monitored NO inacti- vation of PFL-AE by measuring the effect on PFL activation (activity) and UV -vis absorbance changes associated with the essential [Fe 4S4] cluster. To initiate the reaction with NO, 20 μM PFL -AE was mixed with 100 μM NONOate and incubated in a quartz cuvette for 30 min under continuous observation by UV -vis absorption. The reaction was then buffer exchanged to remove residual NO and NONOate by concentrating using 50 kDa MWCO c entrifugal filters and then desalting to buffer consisting of 100 mM Tris, 100 mM KCl at pH 7.6 using a Zeba desalting column. We estimate the final concentration of NO/NONOate after these steps to be <1 μM. The resulting PFL-AE sample was then diluted 50- fold into PFL activation buffer used to activate PFL for 1 h , and PFL activity was measured using the spectrophotomet- ric enzyme-coupled assay described above. A control sam- ple was prepared in the same way in the absence of added NONOate. The i nactivation of PFL- AE was also monitored by EPR spectroscopy. Samples were prepared by mixing 50 μM of enzyme with 500 μM of NONOate in buffer consisting of 100 mM Tris, 100 mM KCl, 10% (w/v) glycerol adjusted to pH 7.6 and incubated at room temperature. W here indicated, samples were also prepared in the same way with the addi- tion of 2 mM SAM or 5 mM NaDT before adding NONOate. The samples were then transferred to EPR tubes and flash frozen in liquid N 2-cooled isopentane (< −130 °C) at differ- ent reaction times and analyzed as previously described. NO T reatment of Bacterial Cell Culture . To observe the effects of NO treatment in the metabolism of E. coli , we in- oculated E. coli BL21 DE3 in 15 mL of LB medium and grew the cells overnight in 50 mL centrifuge tubes in a tube rota- tor at 20 rpm and 37 °C in anaerobic conditions in a vinyl glovebox at < 20 ppm O2. We used 300 μL of these cultures to inoculate 30 mL of M9 medium with 0.4% (w/v) glucose as the only carbon source and grew the cells as previously described. We took 850 μL samples over time and recorded the cell density by OD 600. Each sample was then centrifuged at 20,000 × g for 5 min and the supernatant was used to de- termine the extracellular concentration of the fermentation products lactate, formate, ethanol , and acetate using spec- trophotometric enzyme-coupled assays (Supporting Infor- mation Supplemental Methods). The bacteriostatic and bactericidal effects of NO on anaer- obically growing were assessed during growth in minimal media as previously described. At an OD 600 of 0.35, prior to NO treatment, an aliquot of the cell culture was sampled for cell viability, reported as colony forming units (CFUs), by rapid removal from the anaerobic chamber, centrifugation and serial dilution into sterile Milli -Q water (>17 MΩ) be- fore plating the cell culture on LB -agar plates. After collec- tion of the pre-treatment control the cells were treated with 100 μM NONOate at OD 600 of 0.4 and sampled again at 30 min and 4 h after NO treatment. The plated cells were then incubated at 37 ºC overnight, and colonies were counted the following day, and cell viability was determined as CFU/mL. Similar experiments were performed for cultures that were not treated with NONOate. To analyze the effect of NO over radical species in vivo , and in order to observe the effect of NO on PFL and PFL -AE we transformed E. coli BL21 DE3 with the plasmids pCm8 wt PFL or pCal-n-EK pflA and cultivated the cells overnight in 15 mL of LB medium supplemented with 50 μg/mL of chloramphenicol or kanamycin respectably. We grew the cultures overnight in 50 mL centrifuge tubes in a tube rota- tor at 20 rpm and 37 °C in anaerobic conditions in a vin yl glovebox at < 20 ppm O 2. We used 300 μL of these cultures to inoculate 30 mL cultures of LB supplemented with the corresponding antibiotic. The cells were cultured to OD 600 .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639758doi: bioRxiv preprint of 0.6 and protein expression was induced by adding 1 mM of IPTG. Induced cells were kept growing at 25 ℃ for 18 h , after which 100 μM or 1 mM NONOate was added and incu- bated for 5 min. We proceeded to collect the cells by centrif- ugation at 5 ,000 × g for 5 min and packed the cell pellet in an EPR tube and flash froze them in liquid N 2-cooled isopen- tane. RNR Activation . We activated the class III RNR photo- chemically by mixing either 50 or 100 µM RNR, 5 or 10 µM RNR-AE, 2 mM SAM, 100 µM 5-deazariboflavin in activation buffer composed of 100 mM Tris, 100 mM KCl, 10 mM DTT and 8% (v/v) glycerol at pH 7.6 in a 50 mL beaker, for ki- netic characterization or EPR analysis and NO inhibition, re- spectively. The mixture was exposed to the aforementioned 1 W 405 nm LED light set up for 1 h at room temperature. The extent of activation was estimated by activity assays and EPR quantitation of the resultant G ● relative to a TEMPO standard. Typical activations yielded 0.01 -0.04 G● per dimer. RNR Activity Determined via LC MS Analysis . We measured RNR activity via LC -MS analysis measuring con- version of cytidine triphosphate (CTP) to 2 ′-deoxycytidine triphosphate (dCTP). All activity measurements were per- formed in a VAC Atmosphere glove box (< 2 ppm O 2). Fol- lowing activation of 100 μM RNR, the activated RNR (aRNR) was added to a final concentration of 2 μM aRNR (50 μM to- tal RNR) in assay buffer containing 3 mM CTP, 1 mM ATP, 10 mM formate, 30 mM KCl, 10 mM MgSO4, and 30 mM Tris at pH 7.6. Aliquots were taken from 15 to 300 s and quenched by boiling in a pre-heated 1.5 mL centrifuge tube in a heat block for 2 min. Quenched samples were then brought out of the anaerobic chamber and clarified via cen- trifugation for 15 min at 25,000 × g. 10 μ L of the resulting supernatant was diluted 2 -fold into dephosphorylation buffer with 1 U/mL calf alkaline phosphatase and digested for 2 h at 37 ºC according to the manufacturer’s instructions. Digestion was quenched by addition of 0.1% (v/v) TFA then diluted 20 -fold with Milli -Q water and centrifuged for 30 min at 25,000 × g prior to injection on the LC -MS. Nucleo- side standards of 2 ′-deoxycytosine (dC, 2.5 –50 μM) were prepared in Milli -Q water and adenosine (A, 12.5 μ M) was used as an internal standard. To determine the effect of NO on aRNR activity we acti- vated 100 μ M RNR and treated half of the assay (3.5 μM aRNR) with 500 μ M NONOate, from a 13.6 mM stock solu- tion in 100 mM glycine buffer pH 10 .0, and incubated at room temperature for 10 min. After treatment with NO the samples were immediately diluted 5 -fold in buffer consist- ing of 100 mM glycine at pH 10. 0 to stop the release of NO. The other half of the activation assay was reserved as a con- trol for untreated activity. The quenched NONOate -treated RNR was then diluted 2 -fold into the assay buffer (10 μ M RNR) and activity was measured between 0 s and 5 min as described above. The non-treated control was diluted 5-fold into assay buffer and similarly analyzed for activity from 0 s to 5 min. Assay samples were injected onto a Shimadzu LCMS - 9030 equipped with a Luna polar C18 column (1.6 μ m, 50 mm × 2.1 mm). Nucleosides were resolved with a linear gra- dient composed of mobile phases 0.1% (v/v) TFA in water (C) and 0.1% TFA in acetonitrile (D) from 0 -5% D in C over 8 min with a flow rate of 0.4 mL/min and a column temper- ature of 40 ° C. Single ion monitoring (SIM) was used to quantitate dC (228.1 m/z ± 50 ppm) and A (268.1 m/z ± 50 ppm). Time -dependent dC formation was analyzed using the Shimadzu L ab Solutions Postrun software and deter- mined from the chromatogram SIM MS 1 integrated peak area normalized to the A internal standard and compared to a dC standard calibration curve. EPR of RNR-AE and NikJ Reacted with NO. RNR-AE and NikJ samples for EPR spectroscopy were prepared by mix- ing 50 μM of enzyme with 500 μM of NONOate in buffer con- sisting of 100 mM KCl, 100 mM Tris, and 10% (w/v) glycerol adjusted to pH 7.6 and incubated at room temperature. To examine the role of SAM and reductant on the reactions with NO, samples were prepared in the same way, but with 2 mM SAM, or by preincubating the enzyme for 15 min with 5 mM NaDT before adding NONOate. Samples (250 μL) were then transferred to EPR tubes and flash frozen in liquid N 2- cooled isopentane at different reaction times and analyzed as described above.

Pyruvate Formate Lyase Inactivation by NO. To char- acterize the inactivation of aPFL by NO we used the diethyl- amine diazeniumdiolate (NONOate) as an NO delivery agent, which is a stable aqueous solute at pH > 9, but decom- poses in a pH- and temperature-dependent first order pro- cess.68,69 The release of NO was quantified by monitoring the effectively diffusion-controlled and irreversible conversion of reduced myoglobin (deoxyMb) to the NO-bound myoglo- bin (NO-Mb) via the Soret band shift from 431 nm to 420 nm (Supporting Information Figure S1).70,71 At 30 °C and pH 7.6 we estimated a t1/2 of approximately 25 min, corre- sponding to an NO production rate of 2.05 μM/min and 81 μM/min at 100 μM or 1 mM NONOate, respectfully ( Figure 1, inset). When 10 μM of aPFL is mixed with excess NONOate at either 100 μM or 1 mM and aliquots were sampled for PFL activity, activity was lost concomitantly with NO release (Figure 1). The activity of aPFL followed an exponential de- cay with an apparent rate of 0.26 μM/min at 100 μM and at 2.96 μM/min at 1 mM NONOate. In t he measurement of residual activity of PFL following reaction with NO, enzyme and NONOate were diluted 3,000- fold and free NO was removed by binding to excess deox- yMb. Despite the elimination of NO, the enzyme remained inhibited and showed no signs of recovering over the course of the NO -free activity assay, suggesting the inhibition mechanism is irreversible. For PFL, as with all GREs, elimi- nation of the essential G● abolishes activity, but the enzyme can be re-activated by PFL-AE, assuming no other modifica- tions have been made. To examine the nature of NO inhibi- tion of PFL we completely inactivated aPFL wi th NO, buffer exchanged the inhibited PFL into NO-free reconstitution buffer, and attempted to reactivate the enzyme via the same protocol used to generate the active enzyme initially with PFL-AE. After NO inhibition, the maximal activity that could be recovered varied between 10 -20% (Supporting Infor- mation Figure S2 ). We observe a similar degree of reacti- vation after irreversible inactivation of aPFL by exposure to O 2 (Supporting Information Figure S2), which cleaves the polypeptide chain at the glycine C α, rendering the protein .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639758doi: bioRxiv preprint unactivatable.37,38,72 PFL is proposed to exhibit “half -of- sites” activation, with only one G ● per homodimer , but diradical dimers have not been ruled out .19 As such, we at- tribute the observed inefficient reactivation after treatment with NO to activation of a previously non -activated mono- mer of a former half -of-sites dimer, or to the activation of dimers that had not been activated at all previously. NO Targets the Glycyl Radical of PFL. As a free radical, we expected NO to react with one of the essential amino acid radicals associated with PFL activity, namely the stable G734● (E. coli numbering) or either of the transient thiyl rad- icals of C419 or C418. The G● exhibits a characteristic UV -vis absorption feature at 365 nm that completely decays fol- lowing exposure to NO ( Supporting Information Figure S3). This observation is supported by X-band EPR spectros- copy, where samples were collected before addition of the NONOate and 10 min after ( Figure 2 ). The characteristic asymmetric doublet EPR feature of G 734● is completely lost after 10 min of incubation, consistent with the loss of activ- ity. 22,25 The UV-vis and EPR data are consistent with a mech- anism of inhibition that quenches the endogenous glycyl radical on PFL. Thiyl radicals are known to react with NO to form S -nitrosothiols, 73,74 but alkyl radicals also react with NO, thus the specific radical target of NO inhibition was not obvious.75 We generated C419S and C418S PFL mutants which can be reconstituted to generate G 734●, but are completely inactive, due to the redox-inert nature of serine. While these mutants are not active, they provide insight into the reactiv- ity of NO with PFL. Both C 419S and C 418S mutants showed complete radical loss by EPR over the same time frame as the wt enzyme (Figure 2). To investigate the chemical nature of the product of NO inhibition of aPFL we analyzed the product(s) of aPFL with NO by SDS-PAGE. Samples of aPFL exposed to O 2 are cleaved at the G 734 position, shifting the apparent electrophoretic mobility 3 kDa lower, however samples of aPFL treated with NO do not show changes in the total protein mass (Support- ing Information Figure S4). The lack of change in apparent molecular weight by SDS -PAGE suggests that the chemical modification of aPFL does not involve cleavage of the poly- peptide chain at G 734. We also analyzed tryptic digests of the NO-inhibited PFL by LC -MS/MS for evidence of covalent modifications on peptides containing G734, C419, and C418. De- spite an extensive analysis and the employment of reduct- ants such as DTT, TCEP, and NaBH4, w e observed no evi- dence of C418 or C419 nitrosothiols or G734 nitrosoalkyl/oxime or reduced amine products of any of the radical transfer peptides and no major difference in the MS1 chromatograms between aPFL samples and aPFL samples treated with NO , that could indicate the formation of cross-links between the Figure 1. The inhibition of aPFL by NO. Samples of 10 μM aPFL were mixed with either 100 μM (blue squares) or 1 mM (orange circles) NONOate. Aliquots were taken at the indicated time points and assayed for activity. The activ- ity decay was fit to a single exponential decay model (solid lines). Error bars represent the span of two tech- nical replicates. Inset shows the measured NO released using deoxyMb as a NO reporter and fitted to a linear model (dashed lines). Figure 2. Normalized X-band EPR spectra of aPFL wt and mutants C418S and C419S before (black) and 10 mins after (orange) addition of NONOate. EPR conditions: micro- wave frequency , 9.3 GHz; modulation amplitude , 2 G ; temperature, 100 K. 30 scans were averaged for each spectrum. .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639758doi: bioRxiv preprint modified amino acids and proximal amino acids (Support- ing Information Figure S5). Furthermore, we observed no evidence of a C -terminal oxalylated peptide corresponding to a cleavage of the polypeptide backbone, as seen in the re- action of aPFL with O 2.22 I n all the samples we produced, only the unmodified NH3+-V732SGYAVR738-CO2H tryptic pep- tide was observed. NO inhibits PFL-AE. The EPR spectrum of the NO - inhibited PFL was not completely devoid of EPR -active sig- nals (Figure 2). We suspected the observed residual signal originated from the reaction of NO with PFL -AE, present at 10-fold lower concentration relative to PFL in the activation reaction and carried over into the NO inhibition assay. To investigate this, we analyzed the EPR spectrum of PFL-AE in isolation before and after reacting with NO ( Figure 3). The untreated [Fe 4S4]2+ cluster of PFL-AE is EPR silent, whereas addition of NONOate revealed the formation of an axial EPR signal with prominent features at g⟂ = 2.036 and g∥ = 2.014 (Supporting Information Figure S6A and Table S2). This EPR signature is consistent with previously characterized protein-bound monomeric dinitrosyl iron complexes (DNIC, {Fe(NO)2}9 in Enemark –Feltham notation ) derived from iron -sulfur proteins .76–80 Spin quantification allowed to estimate a rate of DNIC formation of 8.7 μM/min, the re- action is completed in approximately 5 min with an stoichi- ometry of 0.8 spins/mol of PFL -AE ( Supporting Infor- mation Figure S6B and Table S2). The DNIC signal did not change when PFL-AE was provided SAM, suggesting SAM binding does not protect the [Fe 4S4] cluster from decomposition ( Supporting Information Figure S 7 and Table S2). The reaction of PFL-AE and NO was also accom- panied by changes in th e UV-vis spectrum (Supporting In- formation Figure S 8A). We observed a shift in the broad signal associated with the [Fe4S4] cluster with a peak at 410 nm to a new broad signal with a peak at 380 nm , also con- sistent with the monomeric DNIC.81 No further reaction was observed after 15 min (Supporting Information Figure S8B). When in the presence of the reductant NaDT, a differ- ent axial paramagnetic species was formed, with g⟂ = 2.009 and g∥ = 1.970, characteristic of reduced Roussin’s red es- ters ( Supporting Information Figure S 9A and Table S2).78,79 As for the DNIC formation , the Roussin’s red ester signal was not affected by the presence of the substrate SAM (Supporting Information Figure S9B and Table S2). In ei- ther the presence or absence of SAM, the reduction was not complete and roughly 30% of the signal corresponds to a small signal at g = 2.036 remained that we attributed to non- reduced DNIC (Supporting Information Table S2). The [Fe4S4] cluster of PFL-AE is essential for activity, and thus, reaction with NO to form DNICs is expected to inacti- vate the enzyme. Indeed, reacting PFL-AE with NONOate for 30 min inhibits PFL-AE activation of PFL by 99% (Support- ing Information Figure S10). Collectively, the results sup- port a role of NO in degrading the essential [Fe 4S4] cluster of PFL-AE via a protein-bound DNIC intermediate that ren- ders PFL-AE inactive. Figure 3. X-band EPR spectra of 50 μM PFL-AE treated with 500 μ M NONOate freeze-quenched after 30 s (black), 3 mins (gray), and 10 mins (blue) of incubation at room temperature. The 10 min spectrum is plotted vs. magnetic field (B ₀, lower axis ) and g-values are indi- cated. For comparison, the 30 s, 3 min and 10 min spectra are reported vs. g (upper axis, unlabeled). EPR condi- tions: microwave frequency, 9.3 GHz; modulation ampli- tude, 2 G ; power, 2 mW ; temperature, 100 K. 30 scans were averaged for each spectrum. Figure 4. The metabolic impact of NO on anaerobically growing E. coli . Anaerobic cultures of E. coli were sub- jected to treatment with 100 μM NO NOate at mid-expo- nential phase (gray shaded area) . Cell density was esti- mated from the OD600 (black triangles) and extracellular concentration of the fermentation products lactate (blue diamonds), acetate (orange circles) , ethanol (orange squares), and formate (orange triangles) were deter- mined using enzymatic coupled assays. Error bars repre- sent the span between two biological replicates. .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639758doi: bioRxiv preprint Metabolic Consequences of Anaerobic E. coli NO Expo- sure. Having established the in vitro irreversible inactiva- tion of PFL and PFL-AE, we sought to investigate the in vivo consequences of NO treatment on the metabolism of E. coli . We cultivated E. coli cells anaerobically in minimal media with glucose as the unique carbon source, directing glucose metabolism to anaerobic fermentation. C ells in mid- exponential phase were treated with 100 μM NONOate or left unperturbed as a control (Figure 4 and Supporting In- formation Figure S 11). The introduction of NO immedi- ately inhibited cell growth for a period of more than 7 h , demonstrating a bacteriostatic effect of NO in anaerobic conditions. NO also displayed bactericidal effects; cells in the mid-exponential phase displayed a significant decrease in cell viability by 65% within 30 min and 77% after 4 h of NO treatment (Supporting Information Figure S1 2). We also measured the extracellular concentration of the fer- mentation products lactate, formate, acetate , and ethanol using enzyme -coupled assays ( Supporting Information Figure S13). Non-treated cultures produce principally for- mate and lower concentrations of ethanol and acetate, how- ever the addition of NO causes a complete inhibition of the production of ethanol, formate, and acetate and the cell cul- ture accumulated lactate at higher rates and concentrations than in the non- treated cultures. This metabolic shift sug- gests the in vivo inhibition of PFL and PFL-AE by NO. To compare the mechanism of PFL and PFL-AE inhibition in vitro with the potentially more complex chemistry in vivo, we attempted whole-cell EPR of anaerobically grown E. coli overexpressing either PFL or PFL- AE (Figure 5 ). PFL and PFL-AE can be overexpressed in anaerobic conditions and represent > 10% of the total cell protein content , as ob- served by SDS -PAGE ( Supporting Information Figure S14). A fter protein induction and subsequent treatment with NO, we harvested the cells and analyzed them by EPR. E. coli over-expressing PFL can reconstitute the PFL glycyl radical, which can readily be observe by X -band EPR (Fig- ure 5A, inset). The addition of NO quenches the PFL glycyl radical on a timescale similar to the one observed in vitro. We also observed significant DNIC formation in these sam- ples, with characteristic features at g ⟂ = 2.037 and g∥ = 2.015, despite no over -expression of PFL-AE ( Figure 5A , Supporting Information Figure S15, and Table S3). In E. coli cultures over-expressing PFL-AE, a glycyl radical signal can also be observed at lower intensity than the sig- nal observed in samples of the PFL over-expressing cultures (Supporting Information Figure S16A). This signal can be attributed to the genomic PFL expression that can be as high as 20 μM in the cytoplasm of anaerobically growing E. coli. 20,23 However, this glycyl radical signal could corre- spond to other glycyl radical enzymes in the E. coli prote- ome, such as the class III RNR, ketobutyrate formate lyase , YbiW, or PflD.24,82 The [Fe4S4]2+ of PFL-AE is EPR silent be- fore treatment with NO in vivo . Followi ng treatment with 100 μM NONOate, a signal consistent with the formation of DNICs appears with the disappearance of the low -intensity glycyl radical signal ( Figure 5B and Supporting Infor- mation Figure S1 6), and treatment of the cultures with 1 mM of NO NOate produced a composite signal of DNIC and Roussin’s red ester (Supporting Information Figure S16B and Supporting Information Table S3). Non-transformed E. coli cells exposed to NO produce the same signal, although at lower intensity but with similar g-values (Supporting In- formation Figure S17 and Table S3). We attribute this sig- nal to the formation of DNIC s in other iron -sulfur proteins present in the E. coli proteome. NO Inhibits a B road Range of GREs and T heir Acti- vases. Our observation of the NO effect on E. coli Figure 5. X-band EPR spectra of anaerobic E. coli cultures over-expressing A PFL or B PFL-AE before (black) and af- ter (blue and orange, respectively) exposure to 100 μM NONOate for 10 min. Inset in A shows an expanded view of the G ● signal. The asterisk “*” indicates a cavity arti- fact. EPR conditions: microwave frequency, 9.3 GHz; modulation amplitude, 2 G; power, 2 0 μW (A) or 2 mW (B), temperature, 100 K. 30 scans were averaged for each spectrum. .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639758doi: bioRxiv preprint metabolism and loss of the whole-cell glycyl radical EPR sig- nal suggests the in vivo inhibition of PFL. Several enzymes in the glycyl radical enzyme family are associated with pri- mary metabolism in E. coli. Therefore, we sought to investi- gate the reactivity of NO with the E. coli class III RNR and its radical SAM activator enzyme, RNR-AE. The class III RNR is a glycyl radical enzyme found in obligate and facultative anerobic bacteria and archaea, including E. coli , and is es- sential for the anaerobic reduction of nucleotides to deox- ynucleotides for de novo DNA synthesis and repair. 83,84 As with PFL, we examined whether the glycyl radical of RNR reacts with exogenous NO, inhibiting the enzyme. We exposed activated RNR (aRNR) to NO, analogous to our prior experiments with aPFL, and followed the reaction by EPR. After 10 min of treatment with 500 µM NONOate we observed a complete loss of the a RNR glycyl radical signa- ture, as well as the formation of a DNIC signal we attribute to the reaction of remnant RNR -AE with NO from the RNR activation mixture (Figure 6 ). To investigate the effect of glycyl radical loss on the activity of aRNR we utilized LC-MS to quantitate the dephosphorylated reaction product, dC, of CTP reduction ( Supporting Information Figure S1 8 and S19). Non-treated aRNR produced dCTP with a linear rate over 1 min which ultimately slowed as the reaction ap- proached completion; displaying a specific activity of 1,500 ± 100 nmol/min×mg, consistent with literature values (Supporting Information Figure S1 9). 50 On the other hand, aRNR treate d with NONOate for 10 min showed no detectable dC formation over 5 min in the activity assay (<3.5 nmol/min×mg, Figure 6 inset and Supporting Infor- mation Figure S19). To examine the reaction of NO with the RNR-AE in greater detail, we also reacted RNR-AE with NO in isolation and fol- lowed the reaction by EPR. As expected, upon reaction with NO, RNR-AE produced a similar DNIC EPR signal to that ob- served for PFL -AE ( Supporting Information Figure S20A), with similar g⟂ = 2.035 and g∥ = 2.014 (Supporting Information Figure S20B and Table S4) to PFL-AE, and an estimated rate of formation of 16.3 μM/min , again similar to the one observed for PFL -AE (Supporting Information Figure S20C). As for PFL-AE, the DNIC signal of RNR-AE and its g-values were not affected by the presence of the sub- strate SAM (Supporting Information Figure S21 and Ta- ble S 4). In the presence of NO and reducing conditions, RNR-AE formed the characteristic axial paramagnetic signal of Roussin’s red ester, with g⟂ = 2.010 and g∥ = 1.971, and 35% of the signal corresponding to non -reduced DNIC (Supporting Information Figure S 22 and Table S4). The

of both NO inhibition of the class III RNR and the as- sociated RNR-AE suggest a general mechanism of glycyl radical and activator enzyme inhibition by NO. To further establish the generality of inhibition of radical SAM enzyme superfamily members by exogenous NO, we analyzed the inhibition of the enzyme NikJ, a radica l SAM enzyme that catalyzes the C5′ extension from enoylpyruvyl- uridine monophosphate (EP-UMP) to octosyl acid in the bi- osynthesis pathway of nikkomycins. 85,86 PFL-AE and RNR - AE are homologs and share similar function and sequence (sequence identity 28%), while NikJ does not share signifi- cant similarity with either PFL-AE or RNR-AE, representing a distant homolog from the same superfamily. The reaction of NikJ with NO resulted in the formation of an EPR -active DNIC species and followed slower kinetics relative to PFL- AE and RNR-AE (Supporting Information Figure S23 and Table S5). As observed for PFL -AE and RNR -AE the pres- ence of the substrate SAM does not change t he g-values of the observed signal ( Supporting Information Figure S24 and Table S5). NikJ reduced with NaDT produced EPR sig- nals consistent with both a DNIC and Roussin’s red ester (Supporting Information Figure S2 5A and Table S 5), while in the presence of SAM, a signal consistent with Rous- sin’s red ester was observed with nearly identical spectral features as those of PFL-AE and RNR-AE, and showing low concentrations of a non -reduced DNIC signal ( Supporting Information Figure S25B and Table S5).

Phagocytes, including m acrophages, microglia and neu- trophils, along with intestinal epithelial cells , respond to proinflammatory cytokines by expressing the inducible ni- tric oxide synthase (iNOS).87–89 iNOS catalyzes the oxidation of L -arginine into L -citrulline and NO, releas ing high con- centrations of NO into sites of infection . NO acts as both a bactericidal and bacteriostatic agent and its mechanisms of action have been investigated in both in vivo 39,90,91 and in vitro92,93 models. Many molecular targets of NO toxicity have been recognized in bacteria , principally in aerobic condi- tions; NO inhibits DNA replication by targeting DNA - binding zinc metalloproteins 94 and deoxynucleotides pro- duction by reacting with cysteinyl and tyrosyl radical s of RNR.95,96 Additionally, NO impairs respiration by binding to the Cu B of cytochrome bo 97 and central metabolism by Figure 6. X-band EPR spectra of 100 μM reconstituted E. coli class III RNR before (black) and after (blue) treat- ment with 500 μM NONOate for 10 min. The asterisk “*” indicates a cavity artifact. EPR conditions: microwave frequency, 9.3 GHz; modulation amplitude, 2 G; power, 20 μW; temperature, 100 K. 30 scans were averaged for each spectrum. Inset shows dC production by aRNR over 5 min. Single ion monitoring (SIM) LC-MS for dC was per- formed for aRNR (black), aRNR treated with 500 μM NONOate for 10 min (blue), or assay buffer without aRNR (orange). .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639758doi: bioRxiv preprint inhibiting many enzymes in the glycolytic pathway and the TCA cycle.80,98 However, in anaerobic conditions, relevant to gastrointestinal infections, the molecular targets of NO have not been identified with molecular specificity. This knowledge gap limits our understanding of the innate im- mune response in anaerobic environments and hinders the potential application of NO as a therapeutic agent in treating gastrointestinal infections. 17,99 In this study , we identified PFL as a direct target of NO and characterized the mechanism of inhibition. Upon expo- sure to NO, PFL is inhibited with an apparent diffusion-con- trolled rate, suggesting PFL is a kinetically important micro- bial metabolic target of NO . Thiyl radicals react with NO at diffusion-controlled rates,74 however our EPR and site -di- rected mutagenesis data following radical quenching strongly suggest that NO reacts directly with the glycyl rad- ical of the active enzyme. In PFL, C● and G● exist in an equi- librium favoring the formation of G●.100 The lack of evidence of nitrosothiol formation during aPFL inhibition implies a rapid rate of reaction with G● and a low concentration of C● in the G● ⇄ C● equilibrium. Despite our inability to directly demonstrate the nature of the NO adduct(s) to PFL by LC - MS/MS, we hypothesize a radical -radical coupling mecha- nism. The lack of observation of an NO adduct on Gly734 may be related to instability of the modification during MS sam- ple preparation or instability during ionization in the mass spectrometer; such effects have previously been observed for cysteinyl S-NO modified peptides , and their detection generally requires the use of indirect techniques .101–103 To- gether, these data demonstrate that NO is a potent inhibitor of the glycyl radical enzyme PFL via the quenching of the es- sential glycyl radical cofactor. Studies on the effect of NO on anaerobic gastrointestinal bacterial communities indicate that NO exposure influences fermentation products and community composition. 104 To evaluate the effect of PFL inactivation by NO on anaerobic metabolism in a model gastrointestinal microb e, we exam- ined the effect of NO on anaerobic E. coli cell growth, metab- olism, and enzyme post -translational modifications . Using whole-cell EPR, we show that NO reacts with glycyl radicals and FeS clusters in E. coli cultures at a concentration of 100 μM‒comparable to NO levels measured in gastrointestinal samples from patients with inflammatory conditions such as ulcerative colitis and Crohn’s disease. 10,12 Additionally, we observe that NO causes metabolic changes in anaerobic E. coli cultures, arresting the production of the metabolites formate, acetate, and ethanol and halting cell growth and vi- ability. In the absence of PFL activity, lactate accumulates as the product of pyruvate metabolism via lactate dehydrogen- ase, which is not inhibited by NO.39 Similar effects have been reported in anaerobic fermentation from healthy human fe- cal samples treated with NO , wh ere NO produced a long- lasting impact on the metabolome .104 In E. coli, deletion of the pflB gene shifts metabolism to lactate formation, similar to E. coli treated with NO , but does not completely impair growth.105,106 Our observation that growth is effectively halted despite continued lactate production implies a sub- stantial maintenance and repair burden associated with NO exposure. We hypothesized that the inhibition of class III RNR, another glycyl radical enzyme essential to de novo de- oxyribonucleotide synthesis, may c ontribute to the bacteriostatic effect of NO while maintaining lactate fer- mentation. The glycyl radical enzyme , RNR, was indeed in- hibited by NO in vitro, with a reaction rate similar to PFL, suggesting DNA replication and repair are also inhibited, yet further experiments are required to determine the con- sequences of RNR inhibition by NO in vivo. We f urther demonstrate that NO inactivates PFL -AE and RNR-AE. Therefore, NO acts as a general GRE-AE inhibitor, serving a two- fold mechanism of inactivating GRE chemis- try, at both the level of GREs and GRE-AEs. Our EPR data ev- idences the NO reactivity with the enzymes [Fe 4S4] cluster and the formation of DNICs, with similar spectroscopic properties to previously reported iron -sulfur cluster pro- teins and enzymes. 76–80 The DNIC formation is accompanied by a complete loss of activity for PFL-AE, as the [Fe 4S4] is essential for enzyme activity. Radical SAM enzyme iron-sul- fur clusters are coordinated by three cysteines and , when present, the amino and c arboxy group of SAM .27 The pres- ence of SAM provided no protection from NO inhibition for either PFL-AE or RNR -AE, nor the distantly related radical SAM enzyme NikJ, demonstrating that SAM binding and co- ordination of the fourth iron does not protect the iron -sul- fur cluster from decomposition. The collective evidence that GREs and radical SAM en- zymes, including GRE-AEs, are irreversibly inhibited by NO. This suggests that the mechanism by which NO inhibits GREs and GRE -AEs is part of the innate immune response, triggered by gastrointestinal pathogens. Many metabolic pathways in the gut microbiome involve GREs or radical SAM enzymes, indicating that NO effects may be exten- sive. 21,24,27,107–111 Additionally, the enzyme pyruvate:ferre- doxin oxidoreductase is also involved in anaerobic metabo- lism of pyruvate and harbors an iron -sulfur cluster, and is another likely target of anaerobic metabolic inhibition by NO. 112 What remains to be seen is whether the NO response differentially affects the microbiome community, and what defense mechanisms may have adapted to anaerobic NO ex- posure that may inform therapeutic approaches aimed at enhancing the innate immune response. ASSOCIATED CONTENT Supporting Information Supplemental methods, list of primers, UV/vis spectroscopy of myoglobin as an NO sensor , PFL reactivation, aPFL NO inacti- vation followed by UV/vis spectroscopy, Inactivation of aPFL followed by SDS -PAGE, peptide LC -MS/MS of aPFL inhi bited with NO, EPR characterization of PFL-AE and kinetics, EPR sim- ulations parameters for PFL -AE treated with NO, EPR charac- terization of PFL -AE reacted with NO in the presence of SAM, PFL-AE NO inactivation followed by UV/Vis spectroscopy, EPR characterization of reduced PFL-AE with NO, Inhibition of PFL- AE activity by NO, metabolic analysis of anaerobically growing E. coli, metabolites calibration curves, over -expression of PFL and PFL -AE in anaerobic conditions, EPR characterization of whole cells overe xpressing PFL or PFL- AE treated with NO, EPR characterization of RNR- AE NO inactivation, EPR simula- tions parameters for RNR-AE treated with NO, EPR characteri- zation of reduced RNR-AE reacted with NO, deoxycytidine cali- bration curve, LC -MS activity assay o f aRNR and NO treated aRNR, EPR characterization of NikJ NO inactivation, EPR char- acterization of reduced NikJ with NO and EPR simulations pa- rameters for NikJ treated with NO. .CC-BY-NC 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted February 27, 2025. ; https://doi.org/10.1101/2025.02.23.639758doi: bioRxiv preprint AUTHOR INFORMATION Corresponding Author * Brandon L. Greene – Department of Chemistry and Biochem- istry, Interdepartmental Program in Quantitative Biosciences, University of California, Santa Barbara, Santa Barbara, Califor- nia, 93106, United States; Email Address: [email protected] Author Contributions The manuscript was written through contributions of all au- thors. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by the Hellman Faculty Fellowship, a UCSB Faculty Research Grant, and the National Institute of General Medical Sciences of the National Institutes of Health under award number R35GM154993. ACKNOWLEDGMENTS We gratefully acknowledge Prof. Joan Broderick for providing the pCAL-n-EK plasmid. We also acknowledge Dr. Squire Booker for providing t he plasmid pDB1282 and helpful discussion. We also thank Dr. Peter Ford for stimu- lating conversations regarding NO delivery. The plasmid TEVSH was a gift from Helena Berglund (AddGene plasmid #125194). The plasmids pFGET19_Ulp1 and pHYRSF53 were a gift from Hideo Iwai (Addgene plasmid #64697 and #64696). The research reported here used shared facilities of the UC Santa Barbara Materials Research Science and En- gineering Center (MRSEC, NSF DMR-1720256), a member of the Materials Research Facilities Network (http://www.mrfn.org). The Department of Chemistry and Biochemistry Mass Spectrometry Facility instrumentation was supported by the Department of Defense DURIP grant number N00014- 23-1-2197. J.C.C. thanks Anid/Subdirec- ción De Capital Humano/Beca de Doctorado Becas Chile/72200442 for their support. B.L.G thanks the National Institute of General Medical Sciences (R35GM154993), the Hellman Faculty Fellowship , and a UCSB Faculty Research Grant for funding. Additionally, the authors acknowledge the contr ibutions of undergraduate researchers Lindsey Calva, Aisling C. Parast, and Maclean Thomson.

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