Ligand-Induced Heme Ruffling and Bent NO Geometry in Ultra-High-Resolution Structures of Nitrophorin 4 ²,‡ Sue A. Roberts, § Andrzej Weichsel, § Yan Qiu, | John A. Shelnutt, | F. Ann Walker, § and William R. Montfort* ,§ Departments of Biochemistry and Molecular Biophysics, and Chemistry, UniVersity of Arizona, Tucson, Arizona 85721, Biomolecular Materials and Interfaces Department, Sandia National Laboratories, Albuquerque, New Mexico 87185-1349, and Department of Chemistry, UniVersity of New Mexico, Albuquerque, New Mexico 87131 ReceiVed May 7, 2001; ReVised Manuscript ReceiVed August 6, 2001 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 Å) and NH 3 (1.15 Å), 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 Å) and is somewhat bent (Fe-N-O angle ) 156°), 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 Å) and imidazole (1.27 Å). 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. Nitric oxide (NO), 1 a ubiquitous signaling molecule in vertebrates, is synthesized by NO synthase and detected by soluble guanylate cyclase, two heme proteins (recently reviewed in refs 1-4). This creates a challenge for Nature in that NO is reactive toward heme iron and the oxidation state of the heme in these proteins must therefore be carefully regulated. For example, soluble guanylate cyclase uses ferrous [Fe(II)] heme to tightly bind NO (binding affinity ≈ 10 12 M -1 ), which stimulates cyclase activity, but then must somehow release NO in order to turn off the cyclase signal. NO synthase cycles between ferrous and ferric forms while functioning, and apparently suffers from product inhibition by NO. The nitrophorins from Rhodnius prolixus (5) and Cimex lectularius (6) require a ferric heme (binding affinity ≈ 10 6 M -1 ) for storing NO in the salivary gland in a form that can be readily released during feeding. Heme-containing proteins are numerous and participate in a large variety of reactions, including electron transfer (e.g., cytochrome c), transport of gaseous molecules (e.g., hemoglobin), and catalysis (e.g., cytochrome P450). To perform these diverse functions, heme-containing proteins must stabilize appropriate iron oxidation states, provide a range of oxidation potentials for the iron-heme center, and discriminate between ligands. It is clear that the heme rings in proteins are often distorted from planarity and that the type of distortion seen often correlates with protein function (7). Distortions of the heme ring can be caused by protein imposed steric crowding or through covalent linkages between the protein residues and the heme. As the heme ring deviates from planarity, the electronic and chemical proper- ties of the heme iron are altered. Despite these general conclusions, the role of heme distortion in the setting of heme properties is not well characterized, in part due to the sparseness of atomic resolution structures for heme proteins. Nitrophorin 4 (NP4) is one of four NO transport proteins from the saliva of the bloodsucking insect Rhodnius prolixus (recently reviewed in refs 8 and 9). The Rhodnius nitrophor- ins transport NO from the salivary glands of the insects to the tissue of a potential victim, where it is released and induces vasodilation. The proteins also bind tightly to histamine, which is released by the victim in response to the tissue damage caused by the feeding insect. Crystal structures have been determined for three of the four Rhodnius nitrophorins, and all have a lipocalin fold, consist- ² This work was supported in part by National Institutes of Health grants HL62969 to W.R.M and HL54826 to F.A.W. ‡ Coordinates for the NP4-NO, NP4-NH3, NP4-histamine, and NP4-imidazole complexes have been deposited with the Protein Data Bank (PDB entries 1IKH, 1D2U, 1IKE, 1IKJ). * To whom correspondence should be addressed. E-mail: montfort@email.arizona.edu. § University of Arizona. | Sandia National Laboratories and University of New Mexico. 1 Abbreviations: NP1-4, Rhodnius prolixus nitrophorins 1-4; NO, nitric oxide; Fe(II), ferrous iron; Fe(III), ferric iron; K d III , dissociation constant for ligand from ferric nitrophorin; K d II , dissociation constant for ligand from ferrous nitrophorin; OEP, octaethylporphyrinato dianion. 11327 Biochemistry 2001, 40, 11327-11337 10.1021/bi0109257 CCC: $20.00 © 2001 American Chemical Society Published on Web 08/28/2001