title: Kinetics and equilibria in ligand binding by nitrophorins 1-4: evidence for stabilization of a nitric oxide-ferriheme complex through a ligand-induced conformational trap.
abstract: Nitrophorins 1-4 (NP1-4) are ferriheme proteins from the blood-sucking insect Rhodnius prolixus that transport nitric oxide (NO) to the victim, sequester histamine, and inhibit blood coagulation. Here, we report kinetic and thermodynamic analyses for ligand binding by all four proteins and their reduction potentials. All four undergo biphasic association and dissociation reactions with NO. The initial association is fast (1.5-33 microM(-)(1) s(-)(1)) and similar to that of elephant metmyoglobin. However, unlike in metmyoglobin, a slower second phase follows ( approximately 50 s(-)(1)), and the stabilized final complexes are resistant to autoreduction (E degrees = +3 to +154 mV vs normal hydrogen electrode). NO dissociation begins with a slow, pH-dependent step (0.02-1.4 s(-)(1)), followed by a faster phase that is again similar to that of metmyoglobin (3-52 s(-)(1)). The equilibrium dissociation constants are quite small (1-850 nM). NP1 and NP4 display larger release rate constants and smaller association rate constants than NP2 and NP3, leading to values for K(d) that are about 10-fold greater. The results are discussed in light of the recent crystal structures of NP1, NP2, and NP4, which display open, polar distal pockets, and of NP4-NO, which displays an NO-induced conformational change that leads to expulsion of solvent and complete burial of the NO ligand in a now nonpolar distal pocket. Taken together, the results suggest that tighter NO binding in the nitrophorins is due to the trapping of the molecule in a nonpolar distal pocket rather than through formation of particularly strong Fe-NO or hydrogen bonds.
authors.affiliation: Departments of Biochemistry and Chemistry, University of Arizona, Tucson 85721, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Andersen
authors.forename: X D
authors.initials: XD
authors.lastname: Ding
authors.forename: C
authors.initials: C
authors.lastname: Balfour
authors.forename: T K
authors.initials: TK
authors.lastname: Shokhireva
authors.forename: D E
authors.initials: DE
authors.lastname: Champagne
authors.forename: F A
authors.initials: FA
authors.lastname: Walker
authors.forename: W R
authors.initials: WR
authors.lastname: Montfort
mesh_terms: D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006632:Histamine / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D010782:Photolysis; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012239:Rhodnius; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D005290:Ferric Compounds; D006420:Hemeproteins; D008024:Ligands; D011994:Recombinant Proteins; D012471:Salivary Proteins and Peptides; C090560:nitrophorin; D009569:Nitric Oxide; D006632:Histamine