title: 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 approximately 200 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.
authors.affiliation: Department of Biophysics, University of Ulm, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Nienhaus
authors.forename: Estelle M
authors.initials: EM
authors.lastname: Maes
authors.forename: Andrzej
authors.initials: A
authors.lastname: Weichsel
authors.forename: William R
authors.initials: WR
authors.lastname: Montfort
authors.forename: G Ulrich
authors.initials: GU
authors.lastname: Nienhaus
mesh_terms: D000818:Animals; D001665:Binding Sites; D002248:Carbon Monoxide / Q000378:metabolism; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism*; D008024:Ligands; D009569:Nitric Oxide / Q000378:metabolism*; D010777:Photochemistry; D017434:Protein Structure, Tertiary; D012239:Rhodnius; D012471:Salivary Proteins and Peptides / Q000737:chemistry* / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship; D013696:Temperature; D014978:Xenon
chemical_list: D005290:Ferric Compounds; D006420:Hemeproteins; D008024:Ligands; D012471:Salivary Proteins and Peptides; C090560:nitrophorin; D009569:Nitric Oxide; D014978:Xenon; D002248:Carbon Monoxide