NITRIC-OXIDE BINDING AND CRYSTALLIZATION OF RECOMBINANT NITROPHORIN-I, A NITRIC-OXIDE TRANSPORT PROTEIN FROM THE BLOODSUCKING BUG RHODNIUS-PROLIXUS

A nitric Oxide transport protein (nitrophorin I) from the salivary gla nds of the blood-sucking bug Rhodnius prolixus has been expressed as a n insoluble form in Escherichia coli, reconstituted with heme, and cha racterized with respect to NO binding kinetics and equilibria. NO bind ing and absorption spectra for recombinant nitrophorin I were indistin guishable from those of the insect-derived protein. The degree of NO b inding, the rate of NO release, and the Soret absorption maxima for ni trophorin I were all pH dependent. The NO dissociation constant rose 9 -fold over the pH range 5.0-8.3, from 0.19 x 10(-6) to 1.71 x 10(-6). The NO dissociation rate rose 2500-fold between pH 5.0 and pH 8.3, fro m 1.2 x 10(-3) to 3.0 s(-1). Thus, the NO association rate must also b e pH dependent and reduced at pH 5.0 by similar to 280-fold. These fac tors are consistent with nitrophorin function: NO storage in the appar ent low pH of insect salivary glands and NO release into the tissue of the insect's host, where vasodilation is induced. The reversible natu re of NO binding, which does not occur with most other heme proteins, and the apparent kinetic control of NO release are discussed. We also report crystals of nitrophorin I that are suitable for structure deter mination by X-ray crystallography. The most promising crystal form con tains two protein molecules in the asymmetric unit and diffracts beyon d 2.0 Angstrom resolution.