Ligand-Induced Heme Ruffling and Bent NO Geometry in Ultra-High-Resolution Structures of Nitrophorin 4^,

Abstract

The nitrophorins are a family of proteins that use ferric heme to transport nitric oxide (NO) from the salivary glands of blood-sucking insects to their victims, resulting in vasodilation and reduced blood coagulation. We have refined atomic resolution structures of nitrophorin 4 (NP4) from Rhodnius prolixus complexed with NO (1.08 A) and NH(3) (1.15 A), yielding a highly detailed picture of the iron coordination sphere. In NP4-NO, the NO nitrogen is coordinated to iron (Fe-N distance = 1.66 A) and is somewhat bent (Fe-N-O angle = 156 degrees ), with bending occurring in the same plane as the proximal histidine ring. The Fe(NO)(heme)(His) coordination geometry is unusual but consistent with an Fe(III) oxidation state that is stabilized by a highly ruffled heme. Heme ruffling occurs in both structures, apparently due to close contacts between the heme and leucines 123 and 133, but increases on binding NO even though the steric contacts have not changed. We also report the structure of NP4 in complexes with histamine (1.50 A) and imidazole (1.27 A). Unexpectedly, two mobile loops that rearrange to pack against the bound NO in NP4-NO, also rearrange in the NP4-imidazole complex. This conformational change is apparently driven by the nonpolar nature of the NO and imidazole (as bound) ligands. Taken together, the desolvation of the NO binding pocket through a change in protein conformation, and the bending of the NO moiety, possibly through protein-assisted heme ruffling, may lead to a nitrosyl-heme complex that is unusually resistant to autoreduction.

Structural insights into the molecular mechanism of H‐NOX activation

“…5 Biochemical and structural studies have revealed that heme distortion is correlated to changes in heme reactivity, ligand binding, and protein conformation. 5,14,[18][19][20][21]22,23 As observed in the structure reported here, disconnection of the heme and proximal helix in the H102G mutant results in a significantly relaxed heme cofactor conformation. A comparison of the heme cofactors shows that pyrroles A and D move away from the proximal side of the heme pocket toward the distal side in the H102G mutant compared with wild type [ Fig.…”

NO Binding Induced Conformational Changes in a Truncated Hemoglobin from Mycobacterium tuberculosis

“…This conformational change introduces distortions to the heme group and causes the Fe-N-O structure to be bent. It was proposed that this unique heme environment stabilizes the NO and prevents autoreduction of the heme, which is very important for nitrophorin to perform its physiological function (33). Intriguingly, the Fe-NO stretching and Fe-N-O bending frequencies of nitrophorin determined by resonance Raman measurements were identical to those of the NO derivative of HbN (38), despite the fact that the distal pocket of nitrophorin is hydrophobic in contrast to the hydrophilic pocket of HbN (18,19).…”

Spectroscopic identification of reactive porphyrin motions

“…41,42 However, these measurements do not exclude other vibrations that may appear in this frequency range, such as the FeCO distortion mode (in-phase bend/tilt) in iron carbonyl porphyrins [43][44][45] or other heme distortions that may control redox reactions. 46,47 The weak sensitivity of low frequencies to isotopic substitution presents a significant obstacle to associating specific atomic motions with observed frequencies. In contrast, NRVS measurements on oriented single crystals definitively identify Fe motion orthogonal to the heme plane associated with low frequency modes in model compounds.…”