Emxplus spectrometer

Electronic absorption spectra were recorded on a Shimadzu UV-2450 spectrophotometer equipped with a TCC-240A temperature-controlled cell holder. Spectra for WT MbNHase and each variant as well as PtNHase were obtained at 25 °C in a 1 cm quartz cuvette in 50 mM HEPES buffer containing 300 mM NaCl, pH 8.0. X-band EPR spectra for WT MbNHase were recorded at 4 K, 0.1 mW in 50 mM HEPES buffer containing 300 mM NaCl, pH 8.0 on a Bruker EMXplus spectrometer equipped with an ER4116DM (~ 9.6 GHz) resonator, an Oxford Instruments ESR900 helium flow cryostat, and Oxford Instruments ITC503 temperature controller.

The substrate powder (1 mg) was resuspended in H₂O (2.29 mL) and allowed to stir overnight in an anaerobic chamber. Subsequently, the substrate solution (150 μL) was mixed with N₂ purged KPi buffer (50 mm, 350 μL, pH 7.5) and heated for 2 min at 90°C. Immediately after heating, a UV/Vis spectrum of the substrate was recorded that showed a maximum absorbance at λ=307 nm, which confirmed conversion to the enol form. The extinction coefficient (ε₃₀₇=16400m⁻¹ cm⁻¹) was used to calculate the concentration of the final substrate stock used to prepare the enzyme–substrate complexes.
The anaerobically purified protein (300 mm) was thawed, made anaerobic by degassing with N₂ and transferred to a Coy anaerobic chamber. As isolated ARD was transferred directly to a sealed quartz EPR tube and frozen in liquid N₂. Nitrosyl complexes were prepared by the addition of MAHMA NONOate (3 equiv) from a 10 mm stock solution prepared in NaOH (10 mm). The NO-bound enzyme was either transferred to the EPR tube and frozen, or incubated with ACI (10 equiv) prior to freezing. EPR spectroscopy was performed by using an X-band Bruker EMXplus spectrometer equipped with an Oxford Instruments ESR900 liquid helium continuous flow cryostat. Spectra were recorded at 10 K with a 0.6 mT modulation amplitude and 5 mW microwave power.