Resonance Raman spectroscopic study of nitrophorin 1, a nitric oxide-binding heme protein from Rhodnius prolixus, and its nitrosyl and cyano adducts

The resonance Raman (RR) spectra of nitrophorin 1 (NP1) from the saliva of the blood-sucking insect Rhodnius prolixus, in the absence and presence of nitric oxide (NO) and in the presence of cyanide (CN^-), have been studied. The NP1 displayed RR spectra characteristic of six-coordinate high-spin (6cHS) ferric heme at room temperature and six-coordinate low-spin heme (6cLS) at low temperature (77 K). NO and CN^- each bind to Fe^III, both ligands forming 6cLS complexes with NP1. The Fe^III-NO stretching and bending vibrational frequencies of nitrosyl NP1 were identified at 591 and 578 cm^-1, respectively, on the basis of ¹⁵NO isotope shifts. These frequencies are typical of Fe-NO ferric heme proteins, indicating that the NP1 nitrosyl adduct has typical bond strength. Thus, the small NO release rate displayed by NP1 must be due to other protein interactions. Room and cryogenic temperature (77 K) RR spectroscopy and ¹³C, ¹⁵N, and ¹³C¹⁵N isotope substitutions have been used to determine vibrational mode frequencies associated with the Fe^III-CN^- bond for the cyano adducts at 454, 443, 397, and 357 cm^-1. The results were analyzed by normal mode calculations to support the assignment of the modes and to assess the NO and CN^- binding geometries. The observed isotope shifts for the cyano NP1 are smaller than expected and reveal vibrational coupling of Fe^III-CN^- modes with heme modes. We also find that the observed frequencies are consistent with the presence of a nearly linear Fe^IIICN^- linkage (173°) coexisting with a population with a bent structure (155°).