Figure 5.

X-Band EPR spectra of iron–nitrosyl species produced upon addition of NO to the 3mpa-bound (A) and cys-bound (B) enzyme. (A) Comparison of wild-type (3mpa/NO)-bound Av ES-NO relative to H157N and Y159F variants. For the sake of clarity, all spectra are normalized for Fe concentration. For each spectrum, quantitative simulations (dashed lines) are overlaid on the data for comparison. The isotropic $g\sim 2.01$ signal shown in the H157N data is an impurity from addition of methyl viologen and dithionite to the sample. (B) EPR spectra for wild-type (cys/NO)-bound Av ES-NO as compared to an equivalent sample prepared with the Y159F variant. The sharp $g_{\text{ave}}\sim 2.03$ signal in samples prepared with Y159F is attributed to minor dinitrosyl iron species, a common contaminant produced by addition of NO to Fe-containing enzymes.^70–73 The black circles (●) designate the more rhombic (cys/NO)-bound confirmation with observed g-values of 4.31, 3.75, and 1.98. Instrumental parameters: microwave frequency, 9.643 GHz; microwave power, 20 μW; modulation frequency, 0.9 mT; temperature, 10 K. The spectroscopic parameters obtained from data in panels A and B are summarized in Tables 3 and 4.