Table 2.

Hyperfine couplings of weakly coupled ¹⁵N nuclei currently resolved in the (++) quadrant of ¹⁵N HYSCORE spectra of amino acid-specifically nitrogen labeled ARF samples.

Samples/assigned peaks	15Az a (MHz)	15Ay a (MHz)	15Ax a (MHz)	15a b (MHz)	14Az c (MHz)	14Ay c (MHz)	14Ax c (MHz)	14a b (MHz)	References
15N-ARF (uniformly labeled)	1.03	~1.22d	1.1	~1.1d	0.74	~0.9d	0.8	~0.8d	Ref. [35]
	0.43	(0.25)e	0.49	-	0.3	(0.18)e	0.35	-
14N-Tyr/15N-ARFf	1.04	N.R.g	1.12	-	0.74	N.R.g	0.80	-	This work
	0.43		0.49		0.3		0.35
14N-Lys/15N-ARFh, i	0.43	(~0.25)e	0.49	-	0.3	(~0.18)e	0.35	-	This work
15Np1 (Lys45 Nα) peaksj	1.04	0.95	1.10	1.03	0.74	0.68	0.79	0.74	This work

^aHYSCORE spectra recorded at the low- and high-field edges near the maximal and minimal g values give “single-crystal-like” patterns from the reduced Rieske-type cluster, whose g_z and g_x axes are directed along the external magnetic field. In contrast, the resonance condition at the intermediate g_y value is fulfilled by many different, yet well-defined orientations. ¹⁵N hyperfine couplings (¹⁵A_i (i=z,y,x)) are based on a difference of two cross-peak coordinates described in the first-order by the equation: ν_1,2 = |¹⁵ν_N ± ¹⁵A_i/2|. The positions of the peak maxima in the (++) quadrant were determined with the accuracy ~0.04 MHz.

^bA_i = a + T_ii (i=z,y,x), where a is an isotropic hyperfine coupling, and T_ii is an anisotropic hyperfine tensor component which is a diagonal component of the hyperfine tensor in the g-tensor coordinate system; T_xx+T_yy+T_zz=0 due to the tensor properties. Thus, A_x+A_y+A_z=3a and a=(A_z+A_y+A_x)/3. The variations of the splittings at different g_i (i=z,y,x) suggest that the ¹⁵T component of an anisotropic tensor (-T,-T,2T) has an order ~0.1–0.15 MHz (or ¹⁴T ~ 0.07–0.11 MHz), in agreement with the previous considerations based on the point-dipole approximation model.⁴⁷ The tensor component would be larger for nitrogen nuclei located around the innermost Fe(III) side and smaller on the outermost Fe(II) side of the reduced [2Fe-2S] cluster. For the isotropic hyperfine couplings ^14,15a, only the values for the largest splittings are given in this table.

^c14N hyperfine couplings, ¹⁴A_i (i=z,y,x), were recalculated from the corresponding ¹⁵A_i (i=z,y,x) coupling values.

^dThese tentative ^14,15A_y and ^14,15a values were based on the total width of the ¹⁵N HYSCORE spectrum recorded near g_y³⁵ and thus probably overestimated.

^eA very narrow splitting in the (++) quadrant (see Fig. 6B,D).

^fThe ¹⁵N cross-peaks resolved in the (++) quadrant: [2.03, 0.99] and [1.73, 1.30] MHz near g_z; [2.27, 1.15] and [1.94, 1.45] MHz near g_x (see Figs. 5,6).

^gNot resolved (see Fig. 6B).

^hThe ¹⁵N cross-peaks resolved in the (++) quadrant: [1.73, 1.30] MHz near g_z, and [1.94, 1.45] MHz near g_x.

ⁱThe ¹⁴N cross-peaks (¹⁴N_p1) resolved in the (++) quadrant in this sample (see Figs. 4B, 6A,C): [4.08, 3.19] MHz near g_z and [4.41, 3.34] MHz near g_x, which gave the (tentative) ¹⁴N hyperfine couplings ¹⁴A_z of 0.76 MHz near g_z and ¹⁴A_x of 0.87 MHz near g_x, respectively, as estimated using formal expressions for the double-quantum (dq) transitions in the powder-type spectrum, ν_dq± = 2[(¹⁴ν_N ± ¹⁴A_i/2)2 + K² (3 + η²)]^1/2, where K is a nuclear quadrupole coupling constant and η is an asymmetry parameter.

^jThe ¹⁵N_p1 cross-peaks were well-resolved in the difference HYSCORE spectra in the (++) quadrant (see Fig. 6E–G): [2.03, 0.99] MHz near g_z, [2.02, 1.12] MHz near g_y, and [2.24, 1.14] MHz near g_x.