Chemistry & Biology, Volume 21

Supplemental Information

Substrate-Assisted O2 Activation

in a Cofactor-Independent Dioxygenase Sven Thierbach, Nguyen Bui, Josef Zapp, Siri Ram Chhabra, Reinhard Kappl, and Susanne Fetzner

Supplemental Data

Figure S1 (related to Table 1). Organic product of 3-hydroxy-2-methyl-4(1H)-quinolone (MHOQ) con-

version by HodC-W160A.

A: HPLC elution profile of authentic N-acetylanthranilic acid (black trace) and of the product of HodC-

W160A-catalyzed conversion of MHOQ (red trace), detected at 300 nm. B: UV/Vis spectra of the com-

pound eluting at 22 min in the same color code.

Figure S2 (related to Figure 2). Temporal evolution of EPR spectra in the HodC-W160A reaction with 2-

butyl-3-hydroxy-4(1H)-quinolone (BHOQ).

The top spectrum is the first in the time series after starting the reaction, showing the three lines of the 1-

hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) radical CM• and the more intense

signal of the tetrathiatriarylmethyl radical TAM•. Because of the time delay for sequentially recording the

CM• hyperfine lines, the 2

nd and 3

rd line is increasing in intensity during the ongoing reaction. The bottom

graph presents the change in signal intensity for the indicated spectral segment. While the CM• signals are

increasing towards saturation, the TAM signal drops to its minimal intensity when oxygen is completely

consumed.

Figure S3 (related to Figures 3 and 5). Cyclic voltammograms (CV) of CMH and of the organic sub-

strates of the dioxygenase Hod, and pH-dependence.

Graph A shows the CV of 3-hydroxy-2-methyl-4(1H)-quinolone (MHOQ) (olive line) and BHOQ (olive

dashed line), recorded with a scan rate of 0.1 V s–1 at pH 7.8 and 7.4, respectively. The arrows indicate the

first oxidation peak and the less intense reduction peak which were used to estimate the midpoint redox

potential for MHOQ. The very similar voltammograms of MHOQ and BHOQ indicate that the length of

the alkyl group at C-2 has hardly any effect on the electronic properties of the compounds. The CV of the

spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (CMH) (red line) has two well-

developed peak pairs corresponding to the transitions CMH ↔ CM• (W1) and CM

• ↔ CMox (W2). The

response of the buffer is given as the black line. In graph B, the pH dependence of the midpoint potentials

Emid (vs Ag/AgCl) of MHOQ (substrate S) are represented by the olive dots which were fit with a linear

function (olive line). Emid drops linearly by about 75 mV ± 11 mV for an increase of one pH unit over the

pH range 3 to 14, which is consistent with a transfer of a proton and an electron as predicted from the pH-

dependence of the Nernst equation (Bogeski et al., 2011). The two olive dashed lines correspond to the

potential change induced by a 105-fold excess of the reduced or oxidized species, as calculated from the

Nernst equation. [S•]max indicates the one electron oxidized radical species, and [(SH)S

−]max stands for the

protonated or deprotonated reduced substrate MHOQ. The red squares and lines represent the behavior of

transition W1 of CMH with pH, which is quite similar to that of MHOQ (slope of −76 ± 4 mV per pH

unit). The red dashed lines are the 105-fold excess potential limits. The peak pair W2 of CMH is inde-

pendent on pH as expected for the transition CM radical to the oxoammonium state (CMox). The midpoint

potential of O2/O2• −

and the potential change for excess oxygen are given in blue. The black arrow visual-

izes the driving force for the electron transfer from S− to O2 at pH 13, where the potential difference is

about 0.45 V and considerable equilibrium concentrations of S• and superoxide are expected. The gray

arrow indicates the driving force for electron transfer from CMH to a substrate radical with a potential

marked by the x. This process is reflecting the situation in the protein reaction, in which a CMH, sensing

the pH of the buffer, is transferring an electron to the S• located in a high pH environment, thus regaining

the S− substrate state. The S

− state in turn is able to reduce the formed superoxide to peroxide

(Emid(O2• −

/O22−

) = +0.74 V, vs Ag/AgCl), as is described in the main text for reaction sequence (II).

Graph C shows the modeling of the substrate protonation states. The color code of the inset structures

relates them to the pH curves. Using web-based pKa-calculators

(https://ace.chem.illinois.edu/ace/public/pKa.jsp; http://ibmlc2.chem.uga.edu/sparc/), the pH behavior of

MHOQ was simulated yielding pKa values of 9.6 and 11.9, or 10.6 and 13.7. The latter pair is closer to the

result obtained from CV and is modeled in the graph.

Figure S4 (related to Table 2). Thin-layer chromatogram of ethyl acetate extracts of HodC and HodC-

W160A reactions, viewed under UV-light of 366 nm.

For comparability of spot intensities, each sample was re-dissolved in equal amounts of methanol (50 µl),

and TLC plates were spotted with 25 µl of each sample. Symbols indicate N-pentanoylanthranilic acid (■)

and a new compound which shows yellow fluorescence (▲), identified as 2-butyl-2-hydroxy-1,2-

dihydroquinoline-3,4-dione by MS and NMR analyses. Ant, anthranilic acid (bright blue fluorescence).

HPLC analysis of identically prepared samples from HodC-W160A-catalyzed full conversion of 500 µM

BHOQ revealed that ~7 µM of anthranilic acid side product were formed. This suggests that either the 2-

butyl-2-hydroxy-1,2-dihydroquinoline-3,4-dione formed in these reactions decomposes further, or some

decomposition of (an)other intermediate(s) occurs in the HodC-W160A reaction.

Figure S5 (related to Table 2). HMBC (heteronuclear multiple-bond correlation) spectrum of 2-butyl-2-hydroxy-1,2-dihydroquinoline-3,4-dione.

Key correlations are the 3JC,H and

4JC,H couplings between H-5 and the carbonyls C-3 and C-4 and the

2JC,H coupling between the NH proton and the

tertiary alcohol C-2.

Supplemental Experimental Procedures

Synthesis of 2-n-Butyl-3-hydroxy-4(1H)-quinolone (BHOQ)

2-n-Butyl-3-hydroxy-4(1H)-quinolone (BHOQ) was synthesized essentially by the procedure reported by

Ilangovan et al. (2013). The key intermediate 1-chloro-2-hexanone was synthesized by the careful α-

chlorination of the commercially available methyl 3-oxoheptanoate with sulfuryl chloride followed by

acid catalyzed hydrolytic decarboxylation. Esterification of anthranilic acid with 1-chloro-2-hexanone in

N,N-dimethylformamide (DMF) in the presence of anhydrous potassium carbonate gave 2-oxohexyl an-

thranilate. The latter on heating in N-methyl-2-pyrrolidone (NMP) afforded 2-n-butyl-3-hydroxy-4(1H)-

quinolone as a crystalline solid.

Procedure:

Methyl 2-chloro-3-oxoheptanoate: Sulfuryl chloride (3.543 g, 0.0265 mol) was added over 5 min

to a solution of methyl 3-oxoheptanoate (3.995 g, 0.025 mol) in dichloromethane (20 ml) and the mixture

was stirred at room temperature for 2 h. Water (25 ml) was added carefully with cooling and stirred at

room temperature for 15 min. The aqueous was removed and the organic was washed with brine (15 ml),

dried over magnesium sulfate and concentrated to dryness under reduced pressure to give a clear oil

(3.943 g, 81.8 %). 1H NMR (CDCl3) δ 0.95 (3H, t, Me), 1.37 (2H, m, CH2Me), 1.63 (2H, m, CH2CH2Me),

2.73 (2H, m, CH2(CH2)2Me), 3.86 (3H, s, OMe), 4.82 (1H, s, COCHClCO).

1-Chloro-2-hexanone: Methyl 2-chloro-3-oxo-heptanoate (2.0 g, 10.4 mmol), sulfuric acid (4.0 g),

water (6 ml) and dioxane (2 ml) were heated at reflux for a total of 5.5 h. The mixture was cooled, diluted

with water (40 ml) and extracted with dichloromethane (2 × 15 ml). The organic was washed water

(10 ml) and brine (10 ml), dried over magnesium sulfate and concentrated to dryness under reduced pres-

sure to give a brown oil (0.758 g, 54.1 %). 1H NMR (CDCl3) δ 0.94 (3H, t, Me), 1.38 (2H, m, CH2Me),

1.63 (2H, m, CH2CH2Me), 2.62 (2H, m, CH2(CH2)2Me), 4.10 (2H, s, COCH2Cl).

2-Oxohexyl anthranilate: Anhydrous potassium carbonate (0.662 g, 4.8 mmol) was added to a

mixture of anthranilic acid (0.922 g, 6.7 mmol) in DMF (9 ml) and heated at 90 °C for 1 h. The mixture

was cooled to 50 °C and 1-chloro-2-hexanone (0.75 g, 5.6 mmol) was added. The mixture was stirred at

50 °C for 30 min, cooled to room temperature and poured into ice/water (65 ml). The resulting slurry was

stirred at 0 to 10 °C for 1 h before being filtered and washed with water (15 ml) to give a pale brown solid.

(Yield = 1.086 g, 82.5 %).1H NMR (CDCl3) δ 0.94 (3H, t, Me), 1.39 (2H, m, CH2Me), 1.66 (2H, m,

CH2CH2Me), 2.53 (2H, m, CH2(CH2)2Me), 4.86 (2H, s, COCH2CO), 5.7 (1H, b, NH2), 6.67 to 6.71 (2H,

3H & 4H), 7.32 (1H, 5H), 7.97 (1H, 6H).

2-n-Butyl-3-hydroxy-4(1H)-quinolone (BHOQ): 2-Oxohexyl anthranilate (1.0 g, 4.26 mmol) was

heated at reflux in NMP (2.5 ml) for 1 h and then cooled to 50 °C. Ethyl acetate (12 ml) was added and on

cooling to room temperature a cream solid was precipitated. The precipitate was stirred at 0 to 5 °C for

30 min before being filtered and washed with water (3.0 ml), cold ethanol (1.0 ml), and cold ethyl acetate

(1.0 ml) to give the product as a pale brown solid (0.721 g, 78.1 %). 1H NMR (DMSO-d6) δ 0.93 (3H, t,

Me), 1.4 (2H, m, CH2Me), 1.66 (2H, m, CH2CH2Me), 2.74 (2H, t, CH2(CH2)2Me), 7.22 (1H, 8-H), 7.54

(2H, 6-H & 7-H), 8.05 (1H, b, OH) 8.10 (1H, 5-H), 11.42 (1H, s, NH). ES-MS m/z 218.1179 [M+H] +,

C13H16NO2 requires m/z 218.1181).

Other Chemicals

Deferoxamine methanesulfonate (DF), diethyl dithiocarbamate (DETC), the cyclic hydroxylamine CMH,

and the oximetry probe NOX-15.1-OS (a tetrathiatriarylmethyl radical (TAM)), were obtained from Nox-

ygen GmbH (Denzlingen, Germany). Triphenylphosphine (TPP), triphenylphosphine oxide (TPPO), type

VI-A horseradish peroxidase (HRP) and Cu-Zn-superoxide dismutase (SOD) were purchased from Sigma-

Aldrich (Seelze, Germany). Catalase from Aspergillus niger was obtained from Merck (Darmstadt, Ger-

many). Stock solutions of CMH (10 mM) and TAM (5 µM) were prepared in anoxic water containing DF

(25 µM) and DETC (5 µM). Stock solutions of 10 mM TPP and TPPO in methanol were always made

freshly. Stock solutions of 1.0 mg ml–1

HRP were prepared in 100 mM potassium phosphate buffer (pH

6.0) and aliquots were stored at –20 °C.

Protein Expression and Purification, Enzyme Assays

Growth of E. coli M15 [pREP4, pQE30-hodC69S] as well as E. coli harboring pQE30-hodC69S-W160A

or pQE30-hodC69S-H251A, and purification of His6-tagged Hod proteins by affinity chromatography and

anion exchange chromatography were performed as described previously (Beermann et al., 2007). Purified

protein was washed and concentrated in 20 mM potassium phosphate buffer (pH 7.4) containing glycerol

(10% by vol.) by ultrafiltration, and stored at –80 °C. Protein concentrations were determined by absorp-

tion measurements at 280 nm using a NanoDropTM

ND-100 spectrophotometer (PEQLAB, Erlangen,

Germany). Extinction coefficients of 64,525 M–1

cm–1

and 59,025 M–1

cm–1

were calculated for His6HodC

and His6HodC-W160A, respectively. For measuring the activity of Hod proteins, 980 µl of air-saturated

buffer (50 mM Tris/HCl, 2 mM EDTA, pH 8.0), 10 µl of enzyme solution and 10 µl of MHOQ (3-

hydroxy-2-methyl-4(1H)-quinolone) or BHOQ solution (10 mM in ethanol) were mixed in a cuvette and

substrate consumption was monitored at 334 nm or 337 nm (MHOQ: ε334 nm = 9,570 M–1

cm–1

; BHOQ: ε337

nm = 9,556 M–1

cm–1

) in a Jasco V-550 UV/VIS spectrophotometer. 1 unit (1 u) of Hod activity was de-

fined as the amount of enzyme that converts 1 µmol of organic substrate per minute at 30 °C. For the de-

termination of steady-state-kinetic parameters of HodC-W160A protein for MHOQ and BHOQ, the sub-

strate concentrations were varied between 10 µM and 550 µM, and 10 µM and 140 µM, respectively. To

determine the kinetic parameters for O2 (at 100 µM MHOQ), air-saturated enzyme and MHOQ solutions

were mixed with appropriate amounts of N2-, air-, or O2-saturated buffer in an anaerobic cuvette, using

gas-tight syringes, to adjust different O2 concentrations. The apparent kinetic constants for the organic

substrate as well as for O2 were deduced by fitting the data to the Michaelis-Menten equation.

To test whether formate is generated in a possible side reaction, MHOQ and BHOQ (200 µM in

20 mM potassium phosphate buffer, pH 7.4, containing 200 µM DF and 40 µM DETC (buffer I)) were

incubated with 0.2 u ml-1

of HodC or HodC-W160A for 1 h, while shaking at 1000 rpm and 25 °C. Subse-

quently, Candida boidinii formate dehydrogenase (Fluka, ~50 u ml-1

) and NAD+ were added to final con-

centrations of 1 u ml-1

and 2 mM, respectively. Formation of NADH was monitored at 340 nm for 90 min.

Stock solutions of NAD+ were always prepared freshly in 100 mM aqueous KH2PO4 (pH 4.5). Reference

samples containing NAD+ and formate dehydrogenase, with and without substrate, were measured for data

correction. The detection limit of the assay was determined with authentic formic acid. Under the condi-

tions used, 5 µM and 1 µM of formic acid were detected with percentage deviations of 11% and 75%,

respectively.

Site-directed Mutagenesis

Site-directed mutagenesis of the hodC69S gene in order to replace residue W160 of HodC by alanine was

performed using pQE30-hodC69S as template and the mutagenic primer pair 5’-

TCGACGTCGCGCTTGACGGGCATGACGAGAAACG-3’ (W160Afor) and 5’-

CGTCAAGCGCGACGTCGAACAGGCCGTGCGTTCC-3’ (W160Arev); the mismatch bases are under-

lined. The introduction of the mutation into hodC69S was verified by sequencing the plasmid insert and

flanking regions (GATC Biotech AG, Konstanz, Germany).

Detection of Organic Hydroperoxide

Organic hydroperoxides oxidize TPP to TPPO, which can be determined by HPLC in combination with a

fluorescence detector (Nakamura and Maeda, 1991). Due to strong quenching of TPPO fluorescence by

both the organic substrates MHOQ/BHOQ and the N-acylanthranilic acid product, an optical assay to di-

rectly follow the kinetics of TPP oxidation during the enzymatic reaction could not be conducted. For the

quantification of TPPO generated during the enzyme-catalyzed reaction, HodC-W160A or HodC were

diluted to a final activity of 0.2 u ml–1

in buffer I. After addition of 1 mM MHOQ or 100 µM BHOQ and

100 µM TPP, the samples were gently shaken at 1000 rpm and 25 °C until full conversion of the substrate.

To prepare samples for HPLC analysis, protein was precipitated with HCl/methanol and removed by cen-

trifugation for 10 min at 20,000 × g. Supernatants were adjusted to pH 2 and 20% (v/v) methanol and sub-

jected to HPLC analysis. TPPO concentrations were calculated based on a calibration curve of authentic

TPPO. For every sample, a reference mixture without substrate was measured as well to subtract the

background TPPO signal. Freshly prepared TPP stock solutions were always used within 1 hour.

HPLC and TLC

HPLC analysis of the products of MHOQ conversion by HodC-W160A and quantification of an-

thranilic acid were performed on a Eurospher II RP C-18 column (250 × 4 mm), using a linear gradient

(40 min and 20 min, respectively) of 40% to 100% methanol with 1 g l–1

aqueous citric acid at a flow rate

of 0.5 ml min–1

. UV/Vis spectra of the eluting compounds were recorded with a diode array detector (L-

2450 LaChrome Elite®, Merck Hitachi).

TPP oxidation products were separated by HPLC on a Eurospher II RP C-18 column (150 × 3

mm), using a linear gradient (30 min) of 50% to 100% methanol with 0.1% aqueous phosphoric acid at a

flow rate of 0.3 ml min–1

. TPPO was quantified by its emission at 295 nm upon excitation at 235 nm.

Samples to be analyzed by TLC contained 0.2 u ml–1

protein, 500 µM BHOQ and, if present,

100 µM TPP or 600 µM CMH, in buffer I. After 60 min of shaking at 25 °C, organic products were ex-

tracted several times with ice-cold ethyl acetate, dried by solvent evaporation in a vacuum concentrator,

and re-dissolved in methanol. Analytical as well as preparative TLC was performed on Polygram

SilG/UV254 plates (Roth, Karlsruhe, Germany), using a mixture of toluene/acetic acid/1,4-dioxane

(60:16:1.6; by vol.) as solvent. TLC plates were viewed under UV light at 254 nm and at 366 nm. Spots of

an unknown compound were scraped off and extracted in methanol.

Mass Spectrometry (MS)

Positive mode electrospray ionization mass spectrometric analysis of products of BHOQ conversion was

performed on an LXQ Finnigan spectrometer (Thermo Scientific, Dreieich, Germany) with the following

parameters: capillary temperature 300 °C or 250 °C; capillary voltage 4.45 kV. The collision energy in the

MSn mode was set to 17 eV or 30 eV. Solutions of the compounds purified by either preparative TLC or

combined anion exchange/reverse-phase chromatography were directly injected to the mass spectrometer.

NMR Spectroscopy

1H- and

13C-NMR spectroscopy was performed with a Bruker Avance 500 spectrometer equipped with a 5

mm TCI cryoprobe for dilute samples. Dried solid sample, isolated by preparative TLC, was dissolved in

deuterated DMSO and measured at 298 K. The chemical shifts were relative to DMSO at δ 2.50 (1H-

NMR) or DMSO-d6 at δ 39.50 (13

C-NMR) using the standard δ notation in parts per million (ppm). The

2D NMR spectra were recorded as gs-HHCOSY, gs-NOESY, gs-HSQCED and gs-HMBC. Expected

chemical shifts and J-couplings for suggested structures were calculated with the prediction tool of

ACD/ChemSketch 7.00 release.

Electron Paramagnetic Resonance (EPR) Spectroscopy

The solution EPR experiments at 25 °C were performed with a Bruker ESP300e spectrometer equipped

with a ST9010 cavity holding the quartz capillary support of a GTC-III gas-temperature controller (Noxy-

gen). Samples for EPR were prepared in buffer I. The cyclic hydroxylamine CMH was used to monitor the

presence of oxidizing equivalents. For optimal detection of the nitroxide radical (CM•)

formed, the modu-

lation amplitude was 0.1 mT, the microwave power 20 mW and the time constant 20 ms. In control exper-

iments, each component of the EPR reaction assays was tested for a possible oxidation of CMH. The low

temperature (70 K and 20 K) EPR measurements were done with an ELEXSYS E680 spectrometer

(Bruker) using an ESR900 cryostat and an ITC504 temperature controller (Oxford Instruments). Sample

preparation for anoxic measurements was done in an anaerobic glove box, using nitrogen-flushed buffer

and substrate solutions. Protein stock solutions, after being degassed under reduced pressure, were over-

laid with moistened argon gas and equilibrated overnight. Samples were transferred into glass capillaries

which were sealed gas-tight for solution experiments, or frozen in liquid nitrogen inside the glove box in

quartz glass EPR tubes (Wilmad).

To measure the formation of oxidizing species during the Hod reaction, all mixtures in buffer I

were supplemented with 0.6 mM of CMH. Consumption of O2 during the enzymatic reaction was fol-

lowed with the oximetry probe TAM. For experiments with MHOQ (1 mM), 0.5 µM of TAM was used

and the amount of HodC and HodC-W160A in the samples was adjusted to equal activities (0.1 u ml–1

). In

the case of BHOQ, 500 µM of the organic substrate, 1 µM of TAM and 0.2 u ml–1

of protein were used. If

present, the concentration of TPP was 100 µM, and concentrations of catalase and SOD were 2500 u ml–1

and 1000 u ml–1

, respectively. In each case, 50 µl of the respective sample was transferred into a glass

capillary immediately after mixing the solutions, and spectra of accumulating radicals were recorded with

a scan time of 50 s. Each spectrum consists of a 3-line EPR signal of the nitroxide radical CM• and a sin-

gle signal of TAM well separated from the triplet signal (Figure S2). For observing the formation of the

CM-radical and loss of the TAM radical (as an indicator for oxygen consumption), each recorded spec-

trum of the time series was saved with the associated time of the internal computer clock (resolution 1 s).

All individual spectra were then evaluated with the in-house program g-medeia with which the spectra

were sorted according to time, and the peak to peak intensity of a selected line was automatically meas-

ured for all spectra and saved in an input file for Origin8.5 for further processing and presentation. A

standard solution of 100 µM TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl), showing an

identical three-line EPR spectrum, was used to determine the concentrations of CM• in the samples. At

20 mW microwave power and in presence of dissolved oxygen, the TAM signal is partially saturated.

When the oxygen concentration is decreasing, the relaxation is slowed down and signal saturation increas-

es. Eventually oxygen is consumed and the signal intensity is minimal and constant. Relaxation via small

amounts of CMH or TEMPOL radicals was found to be negligible and has to be taken into account above

100 µM concentration of the nitroxide radical.

To quantify the amount of H2O2 released during catalytic turnover of BHOQ by HodC-W160A in

the presence of CMH, an assay was established using CMH as the electron donor for the catalytic reduc-

tion of H2O2 by horse-radish peroxidase (HRP). In the absence of the catalyst, H2O2 (up to 200 µM in the

assay) does not oxidize CMH. Mixtures of HodC-W160A (final activity of 0.2 u ml–1

) and 600 µM CMH

were incubated with 100 µM, 200 µM and 500 µM BHOQ in buffer I at 25 °C, while gently shaking at

1000 rpm. After 30 min, 45 min and 90 min, the amount of CM• was determined by analyzing the corre-

sponding EPR spectra. For measuring the H2O2 content, 25 µl of the same sample were diluted in buffer I

together with another 600 µM CMH. Addition of 100 µg ml–1

HRP (to give a final volume of 50 µl) re-

sulted in complete turnover of H2O2 and formation of CM• by HRP within seconds. From the EPR spectra,

the overall amount of CM• was determined and corrected for the preformed CM

• to give the amount of

H2O2 formed during BHOQ conversion by HodC-W160A. Because control experiments showed some

spontaneous decay of H2O2, the measured H2O2 concentrations had to be corrected. The calculated loss of

H2O2 was 5% within 30 min of incubation.

Cyclic Voltammetry

Cyclic voltammetric (CV) measurements were performed with a PGSTAT12 Potentiostat (Autolab, Neth-

erlands) at 25 ± 1 °C under quiescent condition by using a three-electrode configuration in a one-

compartment cell. A glassy carbon (GC) was used as the working electrode, a saturated silver chloride

Ag/AgCl as the reference electrode and a platinum wire as the auxiliary electrode. All potential values are

therefore given with respect to Ag/AgCl. The potential scan rate was set to 0.1 V s–1

. MHOQ and BHOQ

were dissolved in dimethyl sulfoxide (DMSO), CMH in Millipore water. Measurements were conducted in

deaerated and nitrogen flushed phosphate buffer (50 mM, pH 7.8 or pH 7.4) with final concentrations of

MHOQ or BHOQ and CMH of 0.4 mM and 2 mM, respectively. For pH variation in the range from 3 to

11, phosphate buffers (50 mM) were prepared and adjusted with HCl or NaOH. For pH 12-14 unbuffered

NaOH solutions of appropriate concentrations were used.

Supplemental References

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Stability, unfolding, and structural changes of cofactor-free 1H-3-hydroxy-4-oxoquinaldine 2,4-

dioxygenase. Biochemistry 46, 4241–4249.

Bogeski, I., Kappl, R., Kummerow, C., Gulaboski, R., Hoth, M., and Niemeyer, B.A. (2011) Redox regu-

lation of calcium ion channels: Chemical and physiological aspects. Cell Calcium 50, 407-423.

Ilangovan, A., Fletcher, M., Rampioni, G., Pustelny, C., Rumbaugh, K., Heeb, S., Cámara, M., Truman,

A., Chhabra, S.R., Emsley, J., et al. (2013). Structural basis for native agonist and synthetic inhibitor

recognition by the Pseudomonas aeruginosa quorum sensing regulator PqsR (MvfR). PLoS Pathog. 9,

e1003508.

Nakamura, T., and Maeda, H. (1991) A simple assay for lipid hydroperoxides based on tri-

phenylphosphine oxidation and high-performance liquid chromatography. Lipids 26, 765–768.