Resonance Raman spectroscopic study of nitrophorin 1, a nitric oxide-binding heme protein from Rhodnius prolixus, and its nitrosyl and cyano adducts
Resonance Raman spectroscopic study of nitrophorin 1, a nitric oxide-binding heme protein from Rhodnius prolixus, and its nitrosyl and cyano adducts
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DOI：
10.1021/ja0031927
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发表时间：
2001-11-28
影响因子：
15
通讯作者：
Czernuszewicz, RS
中科院分区：
文献类型：
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作者：
Maes, EM;Walker, FA;Czernuszewicz, RS
研究了吸血昆虫Rhodnius prolixus唾液中硝基磷1 （nitrophorin 1, NP1）在一氧化氮（NO）和氰化物（CN-）存在下的共振拉曼（RR）光谱。NP1在室温下表现出六坐标高自旋铁血红素（6cHS）和低温（77 K）下六坐标低自旋铁血红素（6cLS）的RR光谱特征。NO和CN-分别与Fe-III结合，两种配体都与NP1形成6cLS配合物。根据(NO)-N-15同位素位移，确定了NP1的Fe-III-NO拉伸和弯曲振动频率分别为591和578 cm（-1）。这些频率是Fe-NO铁血红素蛋白的典型频率，表明NP1亚硝基加合物具有典型的键强度。因此，NP1表现出的小NO释放率一定是由于其他蛋白质的相互作用。利用室温和低温（77 K） RR光谱和C-13、N-15和(CN)-C-13-N-15同位素取代测定了氰基加合物在454、443、397和357 cm（-1）处与(FeCN-)- c - iii键相关的振动模式频率。结果通过正常模式计算进行分析，以支持模式分配并评估NO和CN结合几何形状。观察到的氰化物NP1的同位素位移比预期的要小，并且揭示了(FeCN-)- c - iii模式与血红素模式的振动耦合。我们还发现观察到的频率与具有弯曲结构（155度）的种群共存的近线性(FeCN-)- c - iii连锁（173度）的存在一致。
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 (NO)-N-15 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 C-13, N-15, and (CN)-C-13-N-15 isotope substitutions have been used to determine vibrational mode frequencies associated with the (FeCN-)-C-III 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 (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 population with a bent structure (155 degrees).