Structural Dynamics Controls Nitric Oxide Affinity in Nitrophorin 4

Abstract

Nitrophorin 4 (NP4) is one of seven nitric oxide (NO) transporting proteins in the blood-sucking insect Rhodnius prolixus. In its physiological function, NO binds to a ferric iron centered in a highly ruffled heme plane. Carbon monoxide (CO) also binds after reduction of the heme iron. Here we have used Fourier transform infrared spectroscopy at cryogenic temperatures to study CO and NO binding and migration in NP4, complemented by x-ray cryo-crystallography on xenon-containing NP4 crystals to identify cavities that may serve as ligand docking sites. Multiple infrared stretching bands of the heme-bound ligands indicate different active site conformations with varying degrees of hydrophobicity. Narrow infrared stretching bands are observed for photodissociated CO and NO; temperature-derivative spectroscopy shows that these bands are associated with ligand docking sites close to the extremely reactive heme iron. No rebinding from distinct secondary sites was detected, although two xenon binding cavities were observed in the x-ray structure. Photolysis studies at ϳ200 K show efficient NO photoproduct formation in the more hydrophilic, open NP4 conformation. This result suggests that ligand escape is facilitated in this conformation, and blockage of the active site by water hinders immediate reassociation of NO to the ferric iron. In the closed, low-pH conformation, ligand escape from the active site of NP4 is prevented by an extremely reactive heme iron and the absence of secondary ligand docking sites. Nitrophorins (NPs)1 are proteins that transport nitric oxide (NO) from the salivary glands of blood-sucking insects to their victims, resulting in vasodilation and reduced blood coagulation (1-4). Seven nitrophorins (NP1-7) have been identified from Rhodnius prolixus (4 -6), all of which share the lipocalin fold (7) consisting of a ␤-barrel with a highly nonplanar, ruffled heme inserted into one end of the barrel and attached to the protein through a proximal histidine (8). Of the seven Rhodnius nitrophorins, NP4 has been particularly well characterized (10 -13).2 Like all other NPs, NP4 requires a ferric heme to function as an NO-binding protein. Reduction of the heme iron will cause nearly irreversible binding of NO. In the absence of NO, NP4 has a spacious distal pocket that allows easy solvent access (12). Upon NO binding, two loops, the A-B loop (residues 31-37) and the G-H loop (residues 125-132) pack tightly around the NO ligand and form a hydrophobic trap (13). The trapping mechanism utilizes the pH sensitivity of the protein conformation. At pH 5, the approximate pH of the insect saliva, the "closed" conformation is stabilized for the NO complex, whereas at pH 7, the approximate pH of the victim's tissue, the closed conformation is destabilized (2, 14).The kinetics of NO binding and release by NP4 are multiphasic (2, 14), implying static or dynamic active site heterogeneity, which was, however, not apparent in the ultra-high resolution x-ray structure of NP4NO at pH 5.6. In contrast to x-ray diffraction, in...

Influence of Distal Residue B10 on CO Dynamics in Myoglobin and Neuroglobin

“…For ligand rebinding, the change in spectral area is taken proportional to the fraction of ligands that rebind during acquisition of two successive spectra. However, absorption changes can also arise from ligand dynamics within a docking site [50,51], ligand migration to different docking sites [21,62] and conformational changes of the protein [63]. The temperature ramp protocol ensures that rebinding occurs sequentially with respect to the temperature at which the different processes become activated on the time scale of the experiment.…”

Fourier transform infrared spectroscopy study of ligand photodissociation and migration in inducible nitric oxide synthase

“…Upon thermal dissociation from the heme iron, ligands can remain unbound in site B for some time, which increases their probability to escape from the protein. Without this site, they would immediately recombine with the heme iron, as is, e.g ., observed for NO-transporting nitrophorin 35 and modified cytochrome c 55 .…”

“…Interestingly, the NO stretching absorption is also affected by H4B. The band shifts slightly and, in addition, it becomes rather narrow, which is indicative of a more homogeneous active site environment or restricted dynamics of the heme-bound NO due to the bound H4B 35, 59 . In 2005, Rousseau et al .…”

“…The vibrational bands can reveal changes related to ligand relocation to other sites within the protein 18, 29, 32, 33 , rotational motions of the ligand in these sites 25, 34 and protein conformational changes 35 . Often, there are doublets of bands corresponding to opposite orientations of the CO at a particular transient docking site 27, 29, 32, 36– 38 .…”

Nitrophorins and nitrobindins: structure and function

“…The Asp29 and Glu53 residues in the heme distal pocket of RpNPs are too far to coordinate the heme-Fe atom, but could contribute to the negative mid-point potential of the metal center (67). Accordingly, the introduction of a Glu or Asp residue in the heme distal pocket of Pc-Mb at position E7 decreases the value of the midpoint potential by ~200 mV (68). The negative values of the midpoint potential of the heme-Fe atom of Rp-NPs impair the reductive nitrosylation of the metal center (25,58,67,69), in contrast to most of the α-globins (66)(67)(68)70).…”