Spectroscopic and Functional Characterization of Nitrophorin 7 from the Blood-Feeding Insect Rhodnius prolixus Reveals an Important Role of Its Isoform-Specific N-Terminus for Proper Protein Function^†

Nitrophorins (NPs) are a class of NO-transporting and histamine-sequestering heme b proteins that occur in the saliva of the bloodsucking insect Rhodnius prolixus. A detailed study of the newly described member, NP7, is presented herein. NO association constants for NP7 [ (NO)] reveal a drastic change when the pH is varied from 5.5 (reflecting the insect's saliva) to slightly above plasma pH (7.5) (>10⁹ M^-1 → 4.0 × 10⁶ M^-1); thus, the protein promotes the storage of NO in the insect's saliva and its release inside the victim's tissues. In contrast to the other nitrophorins, NP1−4, histamine sequestering cannot be accomplished in vivo due to the low binding constant [ (histamine)] of 10⁵ M^-1 compared to the histamine concentration of 1−10 × 10^-9 M in the blood. A major part of this study deals with the N-terminus, ¹Leu-Pro-Gly-Glu-Cys⁵ of NP7, which is not found in NP1−4. Since NP7 has not been isolated from the insects but was recognized in a cDNA library instead, the N-terminal site of signal peptidase cleavage upon protein secretion was predicted by the program SignalP [Andersen, J. F., Gudderra, N. P., Francischetti, I. M. B., Valenzuela, J. G., and Ribeiro, J. M. C. (2004) Biochemistry 43, 6987−6994]. In marked contrast to wild-type NP7, NP7(Δ1−3) exhibits a very high NO affinity at pH 7.5 [ (NO) ≈ 10⁹ M^-1], suggesting that the release of NO in the plasma cannot efficiently be accomplished by the truncated form. Comparison of the reduction potentials of both constructs by spectroelectrochemistry revealed an average increase of +85 mV for various distal ligands bound to the heme iron when the ¹Leu-Pro-Gly³ peptide was removed. However, ¹H NMR and EPR spectroscopy show that the electronic properties of the Fe^III cofactor are similar in both wild-type NP7 and NP7(Δ1−3). Further, thermal denaturation that revealed a higher stability of wild-type NP7 compared to NP7(Δ1−3), in combination with a homology model based on the NP2 crystal structure (rmsd = 0.39 Å), suggests that interaction of the ¹Leu-Pro-Gly³ peptide with the A−B and/or G−H loops is key for proper protein function.