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. 2014 Aug 23;289(41):28260–28270. doi: 10.1074/jbc.M114.579086

FIGURE 5.

FIGURE 5.

Kinetics of WT EhFdp1 oxidation by O2 or NO. EhFdp1 (about 10 μm in FMN) in 50 mm Tris-HCl, 18% (v/v) glycerol, pH 7.5, was pre-reduced in anaerobic conditions by incubation with 300 μm NADH, 0.3 μm FlRd-Red, and 0.4 μm Rd-D in the presence of 640 nm catalase, and mixed in the stopped-flow apparatus with air-equilibrated buffer (∼240 μm O2) or 200 μm NO. T = 5 °C. Panel A, time-resolved spectra collected after mixing reduced EhFdp1 with air-equilibrated buffer. Thick solid line, oxidized EhFdp1; thick dotted line, reduced EhFdp1; thin lines, selected spectra collected at (bottom to top) 1 ms, 2 ms, 3ms, 4 ms, 5 ms, 20 ms, 100 ms, 0.5 s, and 1 s. Arrow indicates the direction of absorption changes. Panel B, spectra acquired during the reaction of reduced EhFdp1 with 200 μm NO. Thick solid line, oxidized EhFdp1; thick dotted line, reduced EhFdp1; thin lines, selected spectra collected at (bottom to top) 10 ms, 0.75 s, 2 s, 3 s, 4.5 s, 6 s, 8 s, 10 s, 13.7 s, 17.6 s, and 20 s. Arrow indicates the direction of absorption changes. Panel C, time course of the reactions of reduced EhFdp1 with O2 or NO, as obtained by SVD analysis of the data in panels A and B.