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

Abstract

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, an important step for NO-dependent signaling. In the five-coordinate nitrosyl complex, NO can reside on either 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, we characterized the Shewanella oneidensis H-NOX (So H-NOX)-NO complex biophysically under limiting and excess NO conditions. The results show that So H-NOX preferably forms a distal NO species with 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 rebinding of the histidine ligand to the heme.

Figure 1. NO-dependent activation of H-NOX protein

(a,b) Proposed mechanism for NO-dependent activation of H-NOX protein. In the presence of a substoichiometric NO concentration (a), NO initially binds to the distal pocket of H-NOX and forms a six-coordinate complex (state 2). NO binding weakens the Fe(II)–histidine bond, which leads to the cleavage of this bond and results in a five-coordinate distal-bound Fe(II)–NO complex (state 3). If excess NO is present (b), another NO molecule can bind to the proximal pocket (state 4) and form a di-nitrosyl intermediate that then has the potential to form a distal or proximal final five-coordinate Fe(II)–NO complex, depending on which NO molecule dissociates from the di-nitrosyl complex (states 3 or 5, in green). Return of the distal NO complex to the five-coordinate histidyl resting state would be a microscopic reverse of the forward reaction (k₋₂, k₋₁). In the absence of excess NO, returning to the resting state of the proximal complex would be more complicated. The proximal-bound NO could dissociate with the assistance of a solution or protein nucleophile (L), or through a four-coordinate porphyrin to which the proximal histidine would then rebind (state 6). (c) Structure of the So H-NOX with its secondary structure designation and with detailed views of the heme binding pocket for the Fe(II)–unliganded state (PDB: 4U99) and the Fe(II)–NO state (PDB: 4U9B). The distal and proximal sub-domains are defined according to their locations relative to His103. (d) Cysteines in the So H-NOX Fe(II) structure. Cys17 was chosen as the MTSL labeling site.

Figure 2. Distance measurement of Fe(II)–NO to So H-NOX C17-MTSL

(a–c) Electronic absorption spectra of So H-NOX-MTSL treated with substoichiometric NO (a), excess NO with buffer exchange (b) and excess NO without buffer exchange (c). The shoulder at 430 nm indicated the partially bound five-coordinate So H-NOX-MTSL-NO complex. The absorbance at 399 nm indicated formation of five-coordinate NO complexes. The absorbance at ~250 nm indicated incomplete decomposition of DETA NONOate (t_1/2 ~ 56 hours at 25 °C). (d) Q-band EPR spectra with data (black), total simulation (magenta), five-coordinate distal Fe(II)–NO simulation (green), six-coordinate proximal Fe(II)–NO simulation (blue) and minor species (gray). Simulation parameters are the same as in Supplementary Figure 2. The “pump” and “probe” positions are denoted by horizontal (at g = 2.01) and vertical arrows (at g = 2.0165), respectively. Note that the Fe(II)–NO signal in the substoichiometric NO spectrum is relatively weak because not all of the heme-Fe cofactor has NO bound. (e) DEER spectra corresponding to the samples in Figure 2d. Black, data; red, fit. The DEER spectrum of the substoichiometric NO sample shows a clean modulation in the time domain that corresponds to a distance of 2.3 nm. The DEER spectra of the non-buffer-exchanged excess NO sample displays a valley in the spin-echo intensity at 144 ns (arising from the frequency that corresponds to the 2.3 nm distance); however the peak at 272 ns is dampened due to the presence of an additional, lower-frequency contribution that corresponds to a longer distance. (f) Distance distributions corresponding to the DEER spectra in e.

Figure 3. So H-NOX signaling assay with different NO concentrations

(a) Electronic absorption spectra of So H-NOX used in the assay: So H-NOX Fe(II) treated without NO (black line), substoichiometric NO (dashed line) or excess NO (gray line). (b) Measurement of So HK autophosphorylation activity regulated by So H-NOX treated with different NO concentrations. P-values of 0.0075, 0.0182 and 0.3975 were calculated to compare relative kinase autophosphorylation with unliganded/sub-NO, unliganded/excess-NO and sub-NO/excess-NO treated So H-NOX, respectively. The experiment was done in triplicate and data represent mean ± SEM.

Figure 4. Proposed mechanism for NO-dependent activation and deactivation of H-NOX protein

(a) Under substoichiometric NO concentrations, the NO molecule initially binds to the distal pocket of H-NOX and forms a six-coordinate complex (state 2), which is followed by the cleavage of the Fe(II)–histidine bond, resulting in a five-coordinate distal-bound Fe(II)–NO complex as the only product (state 3). (b) Under excess NO concentrations, another NO molecule can bind to the proximal pocket to form a di-nitrosyl intermediate (state 4) which could potentially form either a distal (state 3) or proximal final five-coordinate Fe(II)–NO complex (state 5). This intermediate and the proximal Fe(II)–NO complex (highlighted in yellow) are likely to be kinetically unstable due to the faster reverse rate constants (k₃ < k₋₃, k₄ < k₋₄), making the distal Fe(II)–NO complex (state 3, highlighted in green) the only species under physiological concentration of NO. Dissociation of the distal NO would be accomplished through proximal histidine rebinding. The position of the proximal histidine in the proximal NO complex is known from the crystal structure and noted on in state 5, but is unknown in the distal or proposed di-NO complexes and so is shown as being in motion.