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Regulation of nitric oxide signaling by formation of a distal receptor-ligand complex

The binding of nitric oxide (NO) to the heme cofactor of heme-nitric oxide/oxygen binding (H-NOX) proteins can lead to the dissociation of the heme-ligating histidine residue and yield a five-coordinate nitrosyl complex, which is an important step for NO-dependent signaling. In the five-coordinate nitrosyl complex, NO can reside either on the distal or proximal side of the heme, which could have a profound influence over the lifetime of the in vivo signal. To investigate this central molecular question, the Shewanella oneidensis H-NOX (So H-NOX)–NO complex was biophysically characterized under limiting and excess NO. The results show that So H-NOX preferably forms a distal NO species under both limiting and excess NO. Therefore, signal strength and complex lifetime in vivo will be dictated by the dissociation rate of NO from the distal complex and the return of the histidine ligand to the heme.


Discussion of computational analysis of MTSL rotamers
The conformational dynamics of the MTSL spin label were analyzed using the crystal structure of So H-NOX with NO bound on the proximal face of the heme (PDB code 4U9B). 1 This structure is an appropriate starting point for modeling the spectroscopically detected species because the NO-bound forms of So H-NOX (whether proximal or distal, five-coordinate or six-coordinate) are expected to adopt similar global conformations, with His 103 not ligated to the Fe center.
Moreover, the site to which the spin label is attached (Cys 17) is far removed from the heme and its dynamics are expected to be largely uncoupled to conformational changes associated with ligand binding at the heme Fe center. In order to account for small structural differences between five-and six-coordinate Fe(II)-NO species, we constructed a distal, five-coordinate Fe(II)-NO model by inverting the Fe, N, and O atomic coordinates of the proximal, five-coordinate Fe(II)-NO structure about the center of the heme. A six-coordinate, proximal Fe(II)-NO model was built by translating the Fe(II)-NO unit so that the Fe atom is positioned in the center of the heme (a very minor perturbation that was performed for the sake of completeness).
The relevant coordinates are given in Supplementary For both models, the resulting ensemble of distances can be divided into three distinct groups (Supplementary Fig. 6 and 7): a shorter set (MTSL-Fe ≈ 2.1 nm), an intermediate set (MTSL-Fe ≈ 2.5 nm), and a minor, longer set (MTSL-Fe ≈ 3.0 nm).
The distribution of conformers was highly dependent on the freezing temperature specified in the calculation; as such, we chose the default value of 175 K to approximate the water/glycerol glassing temperature and to make the analysis as unbiased as possible. Neither the shorter nor the longer ensemble of conformations corresponds to the experimentally observed distances, so we focus our discussion on the intermediate set of distances. Regardless, all sets of distances show the same changes upon NO coordination to the distal vs proximal faces (vide infra).
Assuming a spin distribution 2,3 of 0.47 spins on Fe and 0.53 spins on NO, and equal spin distribution on N and O (that is, 0.265 spins on each atom), we obtain a weighted distance distribution centered at 2.4 nm. This compares well with the experimentally determined distance of 2.3 nm. For the proximal, six-coordinate Fe(II)-NO structure, we obtain the following MTSL-atom distances: MTSL-Fe = 2.5 nm, MTSL-N = 2.7 nm, and MTSL-O = 2.75 nm. Assuming a spin distribution 2,3 of 0.21 spins on Fe and 0.78 spins on NO, and equal spin distribution on N and O (that is, 0.39 spins on each atom), we obtain a weighted distance distribution for the MTSL-Fe(II)-NO spin pair centered at 2.7 nm, which is also similar to the experimentally derived distance of 2.8 nm. Thus, the computational results show that the effective MTSL-Fe(II)-NO distance should differ by approximately 0.3 nm between NO bound on the distal and proximal faces of the heme. Finally, we note that the computed distance distributions based on the entire ensemble of computed MTSL rotamers shift by ~0.3 nm between distal-and proximal-NO complexes.

Discussion of factors that affect the analysis of DEER spectra
The aim of the EPR/DEER component of our study is to observe how the Fe(II)-NO speciation changes with increasing NO concentration and to track differences in effective distances between the MTSL spin label and the Fe(II)-NO units. We Given that the Fe, N, and O atoms and the nitroxide spin are not perfectly aligned in the real system, the effect of the different electronic structures on the distance distribution should be significantly lower that what is estimated here. The experimentally observed distance difference is much greater-~0.4 nm (4 Å)-and we therefore conclude that this cannot be attributed to the generation of five-and six-coordinate species with NO bound on the same face of the heme.
Orientation selection: The EPR spectrum of the Fe(II)-NO species is sufficiently broad that the probe pulses excite only certain orientations. Therefore, the effective distances between the five-and six-coordinate species are most reliably compared when the DEER experiments probe similar orientations with respect to the molecular frame. DEER spectra were recorded with the probe pulses positioned at g = 2.0165. This corresponds to orientations along gmin of the five-coordinate species (g = [2.111, 2.024, 2.013]) and gmid of the six-coordinate species (g = [2.094, 2.014, 1.977]). As has been noted elsewhere, 2 the gmin vector in five-coordinate Fe(II)-NO species is most closely aligned with the Fe-N(O) vector, and the gmid vector in sixcoordinate Fe(II)-NO species is also most closely aligned with the Fe-N(O) vector ( Supplementary Fig. 9). In addition, the MTSL spin label is nearly perpendicular to the plane of the heme ( Supplementary Fig. 6), so the interspin vector is also closely aligned with the Fe-N(O) vector. Thus, to a reasonable approximation, the orientations that are probed in the DEER experiments are similarly aligned along the interspin vector for both the five-and the six-coordinate Fe(II)-NO species.
Because the DEER experiments probe similar orientations with respect to the molecular frame, comparison of the two MTSL-Fe(II)-NO distances is appropriate.

Supplementary Tables
Supplementary Table 1