Packer E L, Sweeney W V, Rabinowitz J C
J Biol Chem. 1977 Apr 10;252(7):2245-53.
We have directly assigned the 1H NMR corresponding to the cysteinyl protons, the slowly exchangeable protons, and the aromatic ring protons in the 1H NMR spectrum of Clostridium acidi-urici ferredoxin by isotopic labeling and 13C NMR decoupling techniques. We also show that the resonance pattern in the 8- to 20-ppm (from 2,2-dimethyl-2-sialapentanesulfonic acid) region of the 1H NMR spectra of oxidized Clostridium acidi-urici, Clostridium pasteurianum, Clostridium perfringens, and Peptococcus aerogenes ferredoxins are very similar, and we assign the resonances in this region by analogy with the spectrum of C. acidi-urici ferredoxin. The 1H NMR spectra of the beta protons of the cysteinyl residues of these ferredoxins differ, however, from the 1H NMR spectra of equivalent beta protons of the methylene carbon atoms bonded via a sulfur atom to [4Fe-4S] clusters in synthetic inorganic analogues. In the spectra of the synthetic compounds, the beta protons appear as a single resonance shifted 10 ppm from its unbonded reference position. In the spectra of oxidized clostridial ferredoxins, the cysteinyl beta protons appear as a series of at least eight resolved resonances with shifts that range from 6 to 14 ppm, relative to the free amino acid resonance position. This difference in the spectra of the protein and the synthetic compounds probably results from the fact that the equivalent beta protons of the synthetic compounds are not constrained and are free to rotate and thus assume the same average orientation with respect to the [4Fe-4S] cluster. The shift pattern in the 9- to 14-ppm region is identical in three different clostridial ferredoxins. This suggests that the molecular environments of the corresponding cysteinyl residues are identical. Significant differences in the resonance positions occur, however, in the 14- to 18-ppm region, suggesting that the physical environments of these cysteinyl residues differ. This may reflect differences in the orientation of the corresponding cysteinyl residues relative to the [4Fe-4S] clusters or differences in charge density at the cysteinyl beta protons or both. The slowly exchangeable protons were identified by comparing the 1H NMR spectra of ferredoxins reconstituted in H2O and 2H2O. The remaining resonances in the 8- to 20-ppm region were assigned to each of the 2 tyrosyl residues in C. acidi-urici ferredoxin. This was done by comparing the 1H NMR spectra of C. acidi-urici [(3',5'-2H2)Tyr]ferredoxin and C. acidi-urici [PHE2]ferredoxin with that of C. acidi-urici native ferredoxin.
我们通过同位素标记和¹³C NMR去耦技术，直接确定了尿酸梭菌铁氧化还原蛋白¹H NMR谱中对应于半胱氨酰质子、缓慢交换质子和芳香环质子的¹H NMR信号。我们还表明，氧化型尿酸梭菌、巴氏梭菌、产气荚膜梭菌和产气消化球菌铁氧化还原蛋白的¹H NMR谱在8至20 ppm（相对于2,2 - 二甲基 - 2 - 硅戊烷磺酸）区域的共振模式非常相似，并且我们通过与尿酸梭菌铁氧化还原蛋白的光谱进行类比来确定该区域的共振信号。然而，这些铁氧化还原蛋白半胱氨酰残基的β质子的¹H NMR谱与合成无机类似物中通过硫原子与[4Fe - 4S]簇键合的亚甲基碳原子的等效β质子的¹H NMR谱不同。在合成化合物的光谱中，β质子表现为单个共振信号，相对于其未键合的参考位置位移了10 ppm。在氧化型梭菌铁氧化还原蛋白的光谱中，半胱氨酰β质子表现为一系列至少八个分辨的共振信号，相对于游离氨基酸共振位置，位移范围为6至14 ppm。蛋白质和合成化合物光谱中的这种差异可能是由于合成化合物的等效β质子不受约束，可以自由旋转，因此相对于[4Fe - 4S]簇具有相同的平均取向。在9至14 ppm区域的位移模式在三种不同的梭菌铁氧化还原蛋白中是相同的。这表明相应半胱氨酰残基的分子环境是相同的。然而，在14至18 ppm区域的共振位置出现了显著差异，表明这些半胱氨酰残基的物理环境不同。这可能反映了相应半胱氨酰残基相对于[4Fe - 4S]簇的取向差异，或者半胱氨酰β质子处的电荷密度差异，或者两者都有。通过比较在H₂O和²H₂O中重构的铁氧化还原蛋白的¹H NMR谱，确定了缓慢交换质子。在8至20 ppm区域的其余共振信号被分配给尿酸梭菌铁氧化还原蛋白中的两个酪氨酸残基。这是通过将尿酸梭菌[(3',5'-²H₂)Tyr]铁氧化还原蛋白和尿酸梭菌[PHE2]铁氧化还原蛋白的¹H NMR谱与尿酸梭菌天然铁氧化还原蛋白的¹H NMR谱进行比较来完成的。
