Nitric Oxide Binding to the Ferri- and Ferroheme States of Nitrophorin 1, a Reversible NO-Binding Heme Protein from the Saliva of the Blood-Sucking Insect, Rhodnius prolixus

Abstract

The recombinant NO-binding heme protein, nitrophorin 1 (NP1) from the saliva of the blood-sucking insect, Rhodnius prolixus, has been studied by spectroelectrochemistry, EPR, NMR, and FTIR spectroscopies and X-ray crystallography. It is found that NP1 readily binds NO in solution and in the crystalline state, but the protein is not readily autoreduced by excess NO. Likewise, dithionite is not a very effective reductant of NP1. However, the protein can be photoreduced by illumination with visible light in the presence of excess NO, deazaflavin, and EDTA. Optical spectra of the FeIIINO and FeIINO complexes of NP1 are extremely similar, which makes it difficult to characterize the oxidation state of the NO complex by UV−visible spectroscopy. The reduction potential of NP1 in the absence of NO is ∼300 mV more negative than that of metmyoglobin (metMb). In the presence of NO, the reduction potential shifts ∼+430 mV for NP1−NO, but the reduction potential of metMb−NO cannot be measured for comparison. Based on estimated values of Kd for NP1III−NO, the Kd values for the FeII−NO complex are 20.8 and 80.6 fM at pH 5.5 and 7.5, respectively. The lower driving force for NP1 reduction is qualitatively consistent with the slower rate of autoreduction of NP1−NO; the negative charges surrounding the heme probably also play a role in determining the much slower rate of autoreduction. The N−O stretching frequencies of NP1III−NO and NP1II−NO were measured by FTIR spectroscopy. The values obtained are very typical of other heme−NO stretching frequencies in the two oxidation states: νNO = 1917 and 1904 cm-1 for two species of FeIIINO and 1611 cm-1 for FeIINO; the values of νNO are consistent with 6-coordinate “base-on” heme−NO centers for both oxidation states. The breadths of the IR bands are consistent with the large solvent accessibility of the bound NO of NP1 and also with the possibility of minor dissociation of the protein-provided histidine ligand on the IR time scale. The ratio of the two FeIII−NO species changes with pH and the nature of the buffer. The CO complex of the Fe(II) form of NP1 has νCO = 1960 and 1936 cm-1, again showing the presence of two species. Both NMR and X-ray crystallography show that the protohemin center of NP1 imidazole has a very high preference for a single orientation of the unsymmetrical protoheme moiety. The structure shows the Fe−N−O unit to be quite bent, which is consistent with its being the FeII−NO form of the protein, presumably formed by photoreduction in the X-ray beam. The proximal base, His-59, is clearly coordinated to the iron in the crystalline state and in solution at ambient temperatures, based on FTIR data, but EPR studies of dithionite-reduced samples show that a percentage of the protein has lost the histidine ligand from the FeIINO center in frozen solution.