Figure 4.

NO reductase activity of purified mycobacterial cytochrome bcc-aa₃ supercomplex. Four aliquots of 2.1 µM NO (1.8 μL of 1.8 mM NO each) were sequentially added to degassed buffer (buffer composition as in Figure 1) containing DTT (5 mM), MD (0.26 mM) as the reducing system, and glucose (5 mM) and glucose oxidase (16 units/mL) to scavenge residual O₂ in the 1.5-mL reaction chamber. Then, 30 μL of 10 μM cytochrome bcc-aa₃ (200 nM), pre-reduced with DTT (5 mM) and MD (0.33 mM) in the presence of catalase (520 units/mL) and SOD (60 units/mL), was added to the chamber. The NO decay observed before addition of the supercomplex is likely due to the reaction with the reductants. Addition of pre-reduced cytochrome bcc-aa₃ accelerates the NO decay due to catalytic NO consumption. The initial fast drop in the NO concentration observed just after addition of the supercomplex is likely due at least partly to NO binding to cytochrome bcc-aa₃. Following NO depletion from solution, two aliquots of 4.2 µM NO (3.5 μL of 1.8 mM NO each) were sequentially added and catalytic NO consumption further observed. The activity of bcc-aa₃ at [NO] = 8.4 µM was 2.8 ± 0.5 mol NO (mol bcc-aa₃)⁻¹ min⁻¹. The calculated activity is expressed as mean ± standard deviation.