Effect of mutation of carboxyl side-chain amino acids near the heme on the midpoint potentials and ligand binding constants of nitrophorin 2 and its NO, histamine, and imidazole complexes

Abstract

Nitrophorins (NPs) are a group of NO-carrying heme proteins found in the saliva of a blood-sucking insect from tropical Central and South America, Rhodnius prolixus, the "kissing bug". NO is kept stable for long periods of time by binding it as an axial ligand to a ferriheme center. The fact that the nitrophorins are stabilized as Fe(III)-NO proteins is a unique property because most heme proteins are readily autoreduced by excess NO and bind NO to the Fe(II) heme irreversibly (K(d)s in the picomolar range). In contrast, the nitrophorins, as Fe(III) heme centers, have K(d)s in the micromolar to nanomolar range and thus allow NO to dissociate upon dilution following injection into the tissues of the victim. This NO can cause vasodilation and thereby allow more blood to be transported to the site of the wound. We prepared 13 site-directed mutants of three major nitrophorins, NP2, NP1, and NP4, to investigate the stabilization of the ferric-NO heme center and preservation of reversible binding that facilitates these proteins' NO storage, transport, and release functions. Of the mutations in which Glu and/or Asp were replaced by Ala, most of these carboxyls show a significant role stabilizing Fe(III)-NO over Fe(II)-NO, with buried E53 of NP2 or E55 of NP1 and NP4 being the most important and partially buried D29 of NP2 or D30 of NP4 being second in importance. The pK(a)s of the carboxyl groups studied vary significantly but all are largely deprotonated at pH 7.5 except E124.

Figure 1

The ten residues mutated in this study and their relationship to the position of the heme inside the lipocalin β-barrel of NP2-D1A. The position of the A1 residue is also shown, and a closeup of the two buried residues, D29 and E53, is shown at the right. E55 of NP1 and NP4 and D30 of NP4 are at almost identical positions to E53 and D29 of NP2.

Figure 2

a) Spectroelectrochemical titration of the NO complex of NP2-D1A,D89A at pH 5.5 and 27 °C, and in the inset b) the fit of the data to Eq. (7) is shown. The determined E_m value from the fit to these data at pH 5.5 (51 mV) is plotted in c) along with the E_m values at pH 6.5 and 7.5, for NP2-D1A,D89A(NO). In d) is plotted the E_m values for NP2-D1A(NO) against pH, and in e) is plotted the change in E_m for NP2-D1A,D89A(NO) relative to NP2-D1A (ΔE_m = E_{m(NP2-D1A,D89A)} − E_m(NP2-D1A)), which is fit with a pK_a of ~7.2 (in red). In f) is plotted the change in E_m for NP2-D1A,K127A(NO) relative to NP2-D1A, fit with a pK_a of ~5.4 (in green).

Figure 3

ΔE_m(E_m,(mutant) − E_m,(standard)) for the NO-bound mutants of the NPs at pH 7.5 plotted against the distance of Fe to the closest O atom of the mutated carboxyl side chain or the N of the mutated amino side chain (using the NP2-D1A crystal structure, PDB file 2EU7; NP1, file 2NP1 (distance used is the average of the two molecules in the unit cell); NP4, file 1X8P) with NP2-D1A mutants in blue, NP1 and NP4 mutants in red. These distances are from the ‘best’ crystal structures selected; other structures could have been used but did not have the ideal crystal form or resolution. These are shown by the horizontal line in cyan to represent the divergence in the distance of Fe to closest O in the other crystal structures (Supporting Information Table S4), PDB file 1EUO and 2A3F (NP2 structure at pH 7.7 and 6.5, respectively), and 1X8Q (NP4 structure at pH 5.6 (1X8P is at pH 7.4)), and for NP1 the two distances in each of the two molecules in the unit cell of PDB file 2NP1. The dashed lines represent the boundaries of the region covered for Eq. (6) by the distance-dependent effective dielectric constant of Schutz and Warshel, ε_eff(r_ij), which is given by Eq. (10),– and an effective dielectric constant ε_eff = 20.

Figure 4

The Δ_pHΔE_m(ΔE_m,(pH7.5) − ΔE_m,(pH5.5)) for the NO-bound mutants at pH 7.5 plotted against the distance of Fe to closest O/N of the mutated carboxyl/amine side chain (using the NP2-D1A crystal structure PDB file 2EU7 (NP1 structure, PDB file 2NP1, NP4: PDB file 1X8P) with NP2-D1A mutants in blue, and NP1 and NP4 mutants in red (Table 2, columns b and c). The dashed lines represent the boundaries of the region covered for Eq. (6) by the distance-dependent effective dielectric constant of Schutz and Warshel, ε_eff(r_ij), which is given by Eq. (10),– and an effective dielectric constant ε_eff = 20.

Figure 5

a): E55Q mutant showing hydrogen bonds to Y105 (3.2 Å) and Y17 (2.8 Å). B), c): Wild type protein at pH 7. Two arrangements are seen, most likely representing protonated and unprotonated E55. In b, the arrangement is nearly identical to that for Q55 except for a slight rotation of E55 (0.9 Å for the carbonyl oxygen) and Y17 (1.3 Å for the hydroxyl). In c, F107 rotates substantially (3.8 Å for ring carbon CZ) to allow a solvent molecule to enter and solvate E55, which is probably in the deprotonated state. These two arrangements are discussed elsewhere in more detail.–

Figure 6

Spectrophotometric titration of NP2-D1A,D128A with NO at pH 7.5, 27 °C. The fit of the data to Eq. (8) is shown in the inset, and yields a value of ${log}_{10}{K_f^{\mathit{III}}}_{NO}=8.1\pm 0.1{log}_{10}{\text{M}}^{-1}$ (Table 3).

Figure 7

Spectrophotometric titration of NP2-D1A,D128A with histamine at pH 7.5, 27 °C. The fit of the data to Eq. (8) is shown in the inset, and yields a value of ${log}_{10}{K_f^{\mathit{III}}}_{Hm}=7.1\pm 0.1{log}_{10}{\text{M}}^{-1}$ (Table 4).