Fig 2: Re-constituted nitrate reductase activity. (A) Cartoon representation of the re-constitution of full-length NR activity by combination of the separate NR-Mo-heme and NR-FAD fragments in vitro. (B) Anaerobic NADH:nitrate assay of NR-Mo-heme (NIA1 red, NIA2 black) combined with increasing ratios of NR-FAD fragment. Increasing nitrate reductase activity was observed with increasing ratio of FAD-fragment. (C) Steady-state NADH:nitrate kinetics of re-constituted NIA1 (red) and NIA2 (black) activities.

Fig 3: Nitrate reduction by NR-Mo-heme proteins. (A) Anaerobic Michaelis–Menten kinetics of NIA1-Mo-heme (red) and NIA2-Mo-heme (black) measured with the MV:nitrate assay. (B) Kinetic parameters of multiple batches of NIA1-Mo-heme (red) and NIA2-Mo-heme (black) determined in the MV:nitrate assay. The KM and kcat for NIA1-Mo-heme and NIA2-Mo-heme were compared via unpaired t-test (GraphPad Prism 5). The means ± SEM of n = 33 kinetic series for NIA1-Mo-heme (made with 23 protein batches) and n = 13 kinetic series for NIA2-Mo-heme (eight protein batches used) are shown. p-value: *** < 0.001 < ** < 0.01 < * < 0.05.

Fig 4: Inhibition of nitrite reductase activity by nitrate. Using re-constituted NR-activity, NO production by NIA1 (red) or NIA2 (black) was monitored on the NO analyzer with 400 µM nitrite. Increasing concentrations of nitrate were added simultaneously with the nitrite, and NO production decreased. The % residual activity was fitted with a hyperbolic curve (GraphPad Prism 7) and IC50 and Imax for nitrate determined.