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

Nitrophorins 1-4 (NP1-4) are ferriheme proteins from the blood-sucking inse ct Rhodnius prolixus that transport nitric oxide (NO) to the victim, seques ter histamine, and inhibit blood coagulation. Here, we report kinetic and t hermodynamic analyses for ligand binding by all four proteins and their red uction potentials. All four undergo biphasic association and dissociation r eactions with NO. The initial association is fast (1.5-33 mu M-1 s(-1)) and similar to that of elephant metmyoglobin. However, unlike in metmyoglobin, a slower second phase follows (similar to 50 s(-1)), and the stabilized fi nal complexes are resistant to autoreduction (E degrees = +3 to +154 mV vs normal hydrogen electrode). NO dissociation begins with a slow, pH-dependen t step (0.02-1.4 s(-1)), followed by a faster phase that is again similar t o that of metmyoglobin (3-52 s(-1)). The equilibrium dissociation constants are quite small (1-850 nM). NP1 and NP4 display larger release rate consta nts and smaller association rate constants than NP2 and NP3, leading to val ues for K-d that are about 10-fold greater. The results are discussed in li ght of the recent crystal structures of NP1, NP2, and NP4, which display op en, polar distal pockets, and of NP4-NO, which displays an NO-induced confo rmational. 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 packet rather than through formation of particularly strong Fe-NO or hydrogen bonds.