UV‐visible spectroscopy

Optical spectra were recorded using an Uvikon 941 spectrophotometer in 150‐μL quartz cuvettes. The concentration of nNOS was determined optically from the [CO‐reduced]‐[reduced] difference spectrum using Δε_{444–470 nm} = 76 mm ⁻¹·cm⁻¹ 55.

The stability of Fe^II‐CO complexes of WT nNOSoxy and its mutants was monitored at room temperature (~25 °C) following the absorbance at 443 nm over time in the presence of 10 μm BH₄ and in the absence or presence of 10 mm l‐Arg in 50 mm HEPES buffer pH 7.4. The changes in absorption at 443 nm were then plotted over time, and the percentage of remaining absorbance after 45 min were calculated.

The binding affinities of ImH, substrates l‐Arg and NOHA, and substrate analogues 3–12 for WT and Val567 mutants were determined by perturbation difference spectroscopy 37. The native enzymes (~1–2 μm) were incubated 5 min at 4 °C in 50 mm HEPES buffer pH 7.4 in the presence of 0.5 mm ImH and then equally divided into reference and sample cuvettes. After 2 min at room temperature, increasing concentrations of the studied compounds were added to the sample cuvette and equivalent amounts of buffer were added to the reference cuvette. Apparent dissociation constants (K _s,app) were estimated by plotting the difference in absorbance between peak (~395 nm) and valley (~430 nm) as a function of added substrate or substrate analogue concentrations and fitting the data to a hyperbolic one‐site binding model by Origin (OriginLab Corp., Northampton, MA, USA). The K _s,app for substrates and substrate analogues does not take into account the dissociation constant of ImH. Effective K _s values can be deduced from the K _s,app and the K _s values for ImH, assuming simple competitive binding equilibrium 37.