FIG. 4.

NO generation and nitrite reduction by various types of engineered SO proteins. (A, B) Anaerobic NO generation by Mo-domain (0.25 μM) as a function of nitrite concentration (5 μM–5 mM) in the presence of 5 μM sulfite, at pH 6.5 (A) and 7.4 (B), 37°C, in 20 mM BisTris buffer. (C) Corresponding NO generation rates were calculated from the representative traces shown in (A, B). (D) Anaerobic NO generation from the reaction of nitrite (1 mM) with the Mo-domain, holo-SO, or heme domain ([Mo]=0.25 μM for Mo-domain; [Mo]=0.63 μM for holo-SO; [heme]=0.63 μM) at pH 7.4, 25°C, 20 mM BisTris buffer, with 40 μM PHSN was the reducing substrate. (E) Nitrite (10 μM) reduction is carried out in the anaerobic glove box at room temperature when nitrite reacts with SO+sulfite (open circle, blue), SO+PHSN (dark circle, blue), Mo-domain+sulfite (open square, green), Mo-domain+PHSN (dark square, green), heme domain+PHSN (dark triangle, red), and PHSN (open square, black). Concentrations of sulfite, PHSN, SO, Mo-domain, and heme domain are 50, 50, 10, 10, and 10 μM, respectively. (F) Steady-state kinetics of nitrite reduction by PHSN catalyzed by human holo-SO and the isolated Mo-domain, recorded spectrophotometrically at 520 nm with at least triplicate measurements. 12 μM human holo SO or human SO Mo-domain was mixed with 250 μM phenosafranine and 0–75 mM nitrite. The reaction was started with 250 μM sodium dithionite, and control experiments were performed without the addition of enzyme.