Alignment between predicted H. capsulatum Nor1p (Hc Nor1p) and F. oxysporum P450nor (Fo P450nor). Identical residues are shaded in black. Gray arrowheads mark the start of the two predicted Nor1 proteins. The start of the Nor1(47-450) protein that was characterized in this study is marked with a white arrowhead. Another potential start that was not examined here is marked with a black arrowhead. The cysteine that ligates the heme is marked with a star. The alignment was generated using AlignX from Vector NTI®(Invitrogen).

Electronic absorption spectra of Nor1p(47-450) in various oxidation/ligation states. (A) SDS-PAGE gel analysis of Nor1p(47-450). Protein samples were mixed with SDS loading dye with reductant, boiled and run on a 10–20% Tris-glycine gel. Lane 1, Unstained Protein Molecular Weight Marker (Fermentas); Lane 2, Nor1p (~2 μg); Lane 3, Nor1p (~10 μg). (B) Solid line, ligand-free Fe³⁺-Nor1p; dashed line, NO-bound Fe³⁺-Nor1p. (C) Solid line, dithionite-reduced Fe²⁺-Nor1p; dashed line, CO-bound Fe²⁺-Nor1p.

Activities of purified Nor1p(48-450). (A) Time course of the absorbance change of NADH (~0.13 mM) at 340 nm in the presence of saturated NO. The hatch mark indicates the time when enzyme (~130 nM) was added to the sample. There was no oxidation observed with NADPH. (B) Time course change of NO peak area measured by NOA. DEA-NONOate (1mM) was allowed to react in 100 mM phosphate buffer, pH 7.5, 5 mM NADH for 30 min at 37°C. The reaction sample (2 mL in a 5 mL gas-tight vial) was then cooled to 25°C before enzyme (~130 nM) was added. Headspace (15 μL) was sampled every few minutes. In the absence of enzyme, there was no significant change in the NO concentration (data not shown). (C) Time course change of N₂O peak area measured by GC-ECD. DEA-NONOate (500 μM) was allowed to react with 2 mL NADH/phosphate buffer in a 15 mL gas-tight vial before enzyme (~40 nM) was added to initiate the reaction. Headspace (5 μL) was sampled every few minutes. Each time point represents duplicate samples and the error bars represent ranges.

Electronic absorption spectra of NO-bound Fe³⁺-Nor1p (solid line) and its intermediate NADH reduced product (dotted line) observed at −10°C. The NO and NADH concentrations were both ~100 μM and the amount of Nor1p protein was ~2 μM.

Kinetics of NO consumption by Nor1p. (A) Activity in the presence of increasing concentrations of NO. The data were fit to a Michaelis-Menten equation to yield the estimated K_M and maximum turnover number for NO. (B) Activity in the presence of increasing concentrations of NADH. The data were fit to a Michaelis-Menten equation to yield the estimated K_M for NADH.