Supplementary Data. Supplementary Materials and Methods Measurement of NO formation by ozone-based chemiluminescence. Supplementary References

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1 Supplementary Data Supplementary Materials and Methods Measurement of NO formation by ozone-based chemiluminescence Nitric oxide (NO) production was measured by ozonebased chemiluminescence using a Sievers Nitric Oxide Analyzer (NOA 280i) equipped with a liquid sampling purge system (GE Analytical Instruments). The purge vessel was positioned in line between the NOA and helium carrier gas. NO gas generated in the purge vessel is swept away at a rate of 200 ml/min into the NOA detector. Inside the NOA s gasphase reaction chamber, NO rapidly reacts with ozone (O 3 ), producing electronically excited NO 2 *, which emits a photon of light (hv) when it drops to ground state. Light generated from the reaction of NO and O 3 is then filtered to include only a red wavelength ( > 600 nm) and detected by the photomultiplier tube. The NOA s chemiluminescence signal (mv) directly monitors the rate of NO formation as a function of time. Chemiluminescence intensity, measured as voltage (V), is proportional NO concentration. Data were collected using the high sensitivity (0 1 V) setting, unless indicated as low sensitivity (0 10 V). Sievers NO AnalysisTM Software, Liquid (Version 3.2) was used for data acquisition, at a rate of one data point per 0.25 s. The standard glass purge vessel (15-ml) was used to mix and deoxygenate reagents. The purge vessel s heating jacket was connected to a circulating water bath to control reaction temperature. For enzyme reactions, the reaction buffer (20 mm BisTris or 10 mm phosphate ph 6.5 or 7.4) was maintained in the presence of nitrite for 2 min in the sealed purge vessel to equilibrate the temperature to 37 C and to void oxygen from the reagents before initiating the enzyme reaction. Next, reducing substrate (sulfite or phenosafranine) was injected into the purge vessel through the vessel s septa using a Hamilton syringe. Once a stable baseline was established, sulfite oxidase (SO) was injected into the mixture and the reaction was monitored for several minutes, until the reaction was completed. NO production rates were estimated using a calibration curve. Acidified nitrite was reacted with tri-iodide (I - 3 )to produce stoichiometric quantities of NO. A standard curve was used to convert the raw NOA signal (mv) into NO concentration. Specifically, 50 ll nitrite (ranging from 0 to 1000 pmol) prepared in phosphate buffer (10 mm, ph 7.4) were sequentially injected with a Hamilton syringe into the purging I 3 - solution and the resultant peaks were recorded. The peaks obtained from these injections were then integrated for 0.2 min (peak max 0.1 min) with Origin 8.0 software. The area under the curve (AUC) was plotted against the NO concentration (pmol) and fitted to a linear equation as follows: y = 0.119x. NO generation rate (nmol$s - 1 ) was calculated based on AUC of the highest 12-s NO signals, and analyzed. NO produced from the reaction of SO enzyme with nitrite was calculated using the same method. Traces were smoothed by averaging data in a spanning 2 s. After the peak max was identified, the AUC from time x to time x + 12 s was integrated, which was around the peak of the NO trace curve. The measured NOA response was converted to NO concentration using the linear equation derived from the I 3 - standard curve. The area under the curve (with dimensions of mv$min) was divided by the slope from the standard curve (0.119 mv$min$pmol - 1 NO) to determine the NO concentration. Then, the amount of NO was divided by 12 s, and the amount of protein used during each reaction (mg) and NO generation rates were attained (nmol$s - 1 $mg - 1 ). All the NO generation rates were plotted against the corresponding concentrations of nitrite, and fit to a hyperbolic equation, y = (P 1 x)/(p 2 + x). The values of P 1 and P 2 correspond to m max and K m values in the Michaelis Menten equation, that is, m = V max [S]/(K m + [S]). In the case of single turnover reactions (nonsteady state), a similar fit was used, where the P 1 and P 2 terms represent the apparent m max and K m values, which we refer to as k et (rate of electron transfer) and K d (apparent dissociation constant) to indicate the single turnover nature of the studied reaction. Supplementary References 1. Li H, Kundu TK, and Zweier JL. Characterization of the magnitude and mechanism of aldehyde oxidase-mediated nitric oxide production from nitrite. J Biol Chem 284: , Li H, Samouilov A, Liu X, and Zweier JL. Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrite reduction: evaluation of its role in nitric oxide generation in anoxic tissues. J Biol Chem 276: , Maia LB and Moura JJG. Nitrite reduction by xanthine oxidase family enzymes: a new class of nitrite reductases. J Biol Inorg Chem 16: , 2011.

2 Supplementary Table S1. Kinetic Parameters for NO Generation from Nitrite Reduction Catalyzed by Human and Mouse SO, as Well as Recombinant Mo-Domains Protein/domain ph buffer v max (adjusted, nmol$s - 1 $mg - 1 ) K m (mm) k cat (adjusted, s - 1 ) Reference Human SO (wt) a 7.4 Bis-Tris This work Human SO Mo-domain a 7.4 Bis-Tris This work XO 7.4 Phosphate XO 7.4 Phosphate XO 7.4 Phosphate XO 7.4 Phosphate AO 7.4 Phosphate AO 7.4 Phosphate AO 7.4 Phosphate AO 7.4 Phosphate Protein/domain ph buffer m max (nmol$s - 1 $mg - 1 ) K d (mm) k et (s - 1 ) Reference Human SO (wt) b 6.5 BisTris This work Human SO (wt) b 7.4 BisTris This work Mouse SO (wt) b 6.5 BisTris This work Mouse SO (wt) b 7.4 BisTris This work Human SO Mo-domain b 6.5 BisTris This work Human SO Mo-domain b 7.4 BisTris This work Mouse SO Mo-domain b 6.5 BisTris This work Mouse SO Mo-domain b 7.4 BisTris This work a Reducing substrate was phenosafranine. b Reducing substrate was sulfite. SUPPLEMENTARY FIG. S1. NO generation from nitrite/tri-iodide reaction and DETA-NONOate in a purge or nonpurge system. (A) traces of NO generation from nitrite-i - 3 reaction in the range of 2.5 to 500 pmol nitrite in a purge vessel. (B) traces of NO generation from nitrite-i - 3 reaction in the range of 2.5 to 500 pmol nitrite in a nonpurge vessel. (C) area under curve (AUC) versus NO as traces are shown in (A, B). (D) zoom-in traces of NO generation from DETA-NO in the range of 0.2 to 4 mm in a purge vessel. Inset shows the complete traces. (E) zoom-in traces of NO generation from DETA-NO in the range of 0.2 to 4 mm in a nonpurge vessel. Inset shows the complete traces. (F) AUC versus NO as traces are shown in (D, E).

3 SUPPLEMENTARY FIG. S2. NO generation by human SO holo enzyme as a function of nitrite with phenosafranine as reductant in nonpurge and purge systems. Initial traces of NO generation were measured anaerobically by chemiluminescence in the NO analyzer at ph 7.4, 37 C, 20 mm BisTris buffer in the presence of 0.63 lm human SO, 40 lm phenosafranine, and 20 lm 1 mm nitrite in a nonpurge system (A) and a purge system (B). (C) zoomed-in traces of (A). (D) zoomed-in traces of (B). (E) AUC of first 7-min integration versus nitrite concentration as traces were shown in (A, B).

4 SUPPLEMENTARY FIG. S3. Kinetics of NO generation by human SO holo enzyme as a function of nitrite concentration with phenosafranine as reductant. Initial traces of NO generation were measured anaerobically by chemiluminescence in the NO analyzer at ph 6.5, 37 C, 20 mm BisTris buffer in the presence of 0.63 lm human SO, 40 lm phenosafranine, and 10 lm 2 mm nitrite. NO, nitric oxide; SO, sulfite oxidase.

5 SUPPLEMENTARY FIG. S4. Kinetics of NO generation by human SO holo enzyme as a function of sulfite, and by mouse SO holo enzyme as a function of nitrite or sulfite. Initial rates of NO generation were measured anaerobically by chemiluminescence in the NO analyzer at ph 6.5 and 7.4, 37 C, 20 mm BisTris buffer. (A, B) NO generation rates by 0.63 lm human SO and 1 mm nitrite in the presence of 2 lm 5 mm sulfite at ph 6.5 (A) and at ph 7.4 (B). (C, D) NO generation by 0.32 lm mouse SO and 5 lm sulfite in the presence of 10 lm 1 mm nitrite at ph 6.5. (E, F) NO generation rates by 0.32 lm mouse SO and 5 lm sulfite in the presence of 50 lm 1 mm nitrite at ph 7.4. (G) NO generation rates by 0.32 lm mouse SO and 800 lm nitrite in the presence of 2 lm 4 mm sulfite at ph 6.5. (H) NO generation rates by 0.32 lm mouse SO and 1 mm nitrite in the presence of 2 lm 5 mm sulfite at ph 7.4.

6 SUPPLEMENTARY FIG. S5. Kinetics of NO generation by recombinant human Mo-domain as a function of nitrite in the presence of phenosafranine as reductant. Initial traces and rates of NO generation were measured anaerobically by chemiluminescence in the NO analyzer at ph 6.5, 37 C, in 20 mm BisTris buffer. NO generation traces by 0.25 lm human Mo-domain and 40 lm phenosafranine in the presence of 1 lm 0.4 mm nitrite recorded at the high sensitivity setup (A), and in the presence of mm nitrite at the low sensitivity setup (B).

7 SUPPLEMENTARY FIG. S6. Kinetics of NO generation by recombinant human Mo-domain or by mouse Modomain as a function of nitrite or sulfite concentrations. Initial rates of NO generation were measured anaerobically by chemiluminescence in the NO analyzer at ph 6.5 and 7.4, 37 C, in 20 mm BisTris buffer (A F) or in 10 mm phosphate buffer (G, H). (A, B) NO generation rates by 0.25 lm human Mo-domain and 1 mm nitrite in the presence of 2 lm 5mM sulfite at ph 6.5 and 7.4. (C, D) NO generation by 0.25 lm mouse Mo-domain and 5 lm sulfite in the presence of 2.5 lm 10 mm nitrite at ph 6.5. (E, F) NO generation by 0.25 lm mouse Mo-domain and 5 lm sulfite in the presence of 5 lm 10 mm nitrite at ph 7.4. (G, H) NO generation by 0.25 lm mouse Mo-domain and 5 lm sulfite in the presence of 10 lm 1 mm nitrite at ph 6.5 in 10 mm phosphate buffer.

8 SUPPLEMENTARY FIG. S7. Kinetics of NO generation from mouse Mo-domain as a function of sulfite concentration. Initial rates of NO generation were measured anaerobically by chemiluminescence in the NO analyzer at 37 C. (A) NO generation by 0.25 lm mouse Mo-domain and 1 mm nitrite in the presence of 2 lm 5 mm sulfite at ph 6.5, 20 mm BisTris buffer. (B) NO generation rates by 0.25 lm mouse Mo-domain and 1 mm nitrite in the presence of 2 lm 8 mm sulfite at ph 7.4, 20 mm BisTris buffer. (C) NO generation rates by 0.25 lm mouse Mo-domain and 1mM nitrite in the presence of 2 lm 8 mm sulfite at ph 6.5, in 10 mm phosphate buffer. SUPPLEMENTARY FIG. S8. NO generation by mouse SO proteins. Initial rates of NO generation were measured by chemiluminescence in the NO analyzer at ph 6.5, 37 C, in 20 mm BisTris buffer anaerobically. (A) NO generation by 0.25 lm Mo-domain of mouse SO and sulfite (5 lm) in the presence of 2.5 lm 1 mm nitrite. (B) NO generation by 0.38 lm H119A/H144A and sulfite (5 lm) in the presence of 5 lm 1 mm nitrite. (C) NO generation by 0.32 lm mouse SO in the presence of 10 lm 1 mm nitrite.

9 SUPPLEMENTARY FIG. S9. NO generation by human holo SO and human SO Mo-domain aerobically and anaerobically. Initial rates of NO generation were measured by chemiluminescence in the NO analyzer at ph 7.4, 37 C, in 20 mm BisTris buffer anaerobically. (A) Traces of NO generation by human holo SO aerobically and anaerobically when nitrite is 0.1, 1, and 10 mm. (B) NO generation rates were measured as shown in (A). (C) Traces of NO generation by human SO Mo-domain aerobically and anaerobically when nitrite is 0.1, 1, and 10 mm. (D) NO generation rates were measured as shown in (C).