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 (6c HS) 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 fo rming 6cLS complexes with NP1. The Fe-III-NO stretching and bending vibrati onal frequencies of nitrosyl NP1 were identified at 591 and 578 cm(-1), res pectively, on the basis of (NO)-N-15 isotope shifts. These frequencies are typical of Fe-NO ferric heme proteins, indicating that the NP1 nitrosyl add uct has typical bond strength. Thus, the small NO release rate displayed by NP1 must be due to other protein interactions. Room and cryogenic temperat ure (77 K) RR spectroscopy and C-13, N-15, and (CN)-C-13-N-15 isotope subst itutions have been used to determine vibrational mode frequencies associate d with the (FeCN-)-C-III bond for the cyano adducts at 454, 443, 397, and 3 57 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 geometrie s. The observed isotope shifts for the cyano NP1 are smaller than expected and reveal vibrational coupling of (FeCN-)-C-III modes with heme modes. We also find that the observed frequencies are consistent with the presence of a nearly linear (FeCN-)-C-III linkage (173 degrees) coexisting with a popu lation with a bent structure (155 degrees).