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

The recombinant NO-binding heme protein, nitrophorin 1 (NP1) from the saliv a of the bloodsucking insect, Rhodnius prolixus, has been studied by spectr oelectrochemistry, EPR, NMR, and FTIR spectroscopies and X-ray crystallogra phy. It is found that NP1 readily binds NO in solution and in the crystalli ne 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 (FeNO)-N-III and ( FeNO)-N-II complexes of NP1 are extremely similar, which makes it difficult to characterize the oxidation state of the NO complex by UV-visible spectr oscopy. The reduction potential of NP1 in the absence of NO is similar to 3 00 mV more negative than that of metmyoglobin (metMb). In the presence of N O, the reduction potential shifts similar to+430 mV for NP1-NO, but the red uction potential of metMb-NO cannot be measured for comparison. Based on es timated values of K-d for NP1(III)-NO, the K-d values for the Fe-II-NO comp lex 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 o f autoreduction of NP1-NO; the negative charges surrounding the heme probab ly also play a role in determining the much slower rate of autoreduction. T he N-O stretching frequencies of NP1(III)-NO and NP1(II)-NO were measured b y FTIR spectroscopy. The values obtained are very typical of other heme-NO stretching frequencies in the two oxidation states: upsilon(NO) = 1917 and 1904 cm(-1) for two species of (FeNO)-N-III and 1611 cm(-1) for Fe-II-NO; t he values of upsilon(NO) are consistent with 6-coordinate "base-on" heme-NO centers for both oxidation states. The breadths of the IR bands are consis tent with the large solvent accessibility of the bound NO of NP1 and also w ith the possibility of minor dissociation of the protein-provided histidine ligand on the IR time scale. The ratio of the two Fe-III-NO species change s with pH and the nature of the buffer. The CO complex of the Fe(II) form o f NP1 has upsilon(CO) = 1960 and 1936 cm(-1), again showing the presence of two species. Both NMR and X-ray crystallography show that the protohemin c enter 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 Fe-II-NO form of t he protein, presumably formed by photoreduction in the X-ray beam. The prox imal base, His-59, is clearly coordinated to the iron in the crystalline st ate and in solution at ambient temperatures, based on FTIR data, but EPR st udies of dithionite-reduced samples show that a percentage of the protein h as lost the histidine ligand from the (FeNO)-N-II center in frozen solution .