Resonance Raman Spectroscopic Study of Nitrophorin 1, a Nitric Oxide-Binding Heme Protein from Rhodnius prolixus, and Its Nitrosyl and Cyano Adducts

Abstract

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(III)CN(-) linkage (173 degrees ) coexisting with a population with a bent structure (155 degrees ).

Redox-dependent Ligand Switching in a Sensory Heme-binding GAF Domain of the Cyanobacterium Nostoc sp. PCC7120

“…2 and 10 ) are also consistent with a 6cLS heme (39). The CϭC modes characteristic of the heme vinyl substituents are well separated (1615 and 1627 cm Ϫ1 ), indicating a rather different environment of the vinyl groups of rings A and B (36,40).…”

NO Binding Induced Conformational Changes in a Truncated Hemoglobin from Mycobacterium tuberculosis

“…It was proposed that this unique heme environment stabilizes the NO and prevents autoreduction of the heme, which is very important for nitrophorin to perform its physiological function (33). Intriguingly, the Fe-NO stretching and Fe-N-O bending frequencies of nitrophorin determined by resonance Raman measurements were identical to those of the NO derivative of HbN (38), despite the fact that the distal pocket of nitrophorin is hydrophobic in contrast to the hydrophilic pocket of HbN (18,19). In contrast, horse heart myoglobin, which has a distal His at the E7 position that stabilizes the heme-bound NO, has a much more intense Fe-N-O bending mode with a lower frequency (572 cm -1 ) and a higher Fe-NO stretching frequency (596 cm -1 ) (39).…”

Geometries and Electronic Structures of Cyanide Adducts of the Non-Heme Iron Active Site of Superoxide Reductases: Vibrational and ENDOR Studies

“…CN, 13 CN, C 15 N and 13 C 15 N) has been employed to identify these modes and assess the Fe-C-N angle (29)(30)(31)(32)(33)(34)(35)(36)(37)(38). In an "essentially" linear Fe-C-N complex (∼175°), where the bending mode is nonetheless allowed, the resonance Raman spectrum typically exhibits a "monotonic-isotope-shifting" ν(Fe-CN) stretching mode with a frequency 30−60 cm −1 higher than the lower energy "zigzag-isotope-shifting" δ(Fe-C-N) bending mode (30,36,38). A monotonically-isotope-shifting mode exhibits cyanide isotope shifts that vary as a function of total cyanide mass, with the 13 C 14 N and 12 C 15 N isotopomers having similar frequencies, and is characteristic of the ν(Fe-CN) stretching mode of a near-linear Fe-C-N unit A zigzag-isotope-shifting mode exhibits isotope shifts that primarily vary with isotopic substitution of the carbon atom, and thus move in a "zigzag" fashion with the isotopic sequence CN − , 13 CN, C 15 N and 13 C 15 N; this zigzag isotope shifting is characteristic of a mode primarily composed of δ(Fe-C-N) bending character.…”