Taboy C H, Vaughan K G, Mietzner T A, Aisen P, Crumbliss A L
Department of Chemistry, Duke University, Durham, North Carolina 27708, USA.
J Biol Chem. 2001 Jan 26;276(4):2719-24. doi: 10.1074/jbc.M004763200. Epub 2000 Oct 11.
The Fe(3+) binding site of recombinant nFbp, a ferric-binding protein found in the periplasmic space of pathogenic Neisseria, has been characterized by physicochemical techniques. An effective Fe(3+) binding constant in the presence of 350 microm phosphate at pH 6.5 and 25 degrees C was determined as 2.4 x 10(18) m(-1). EPR spectra for the recombinant Fe(3+)nFbp gave g' = 4.3 and 9 signals characteristic of high spin Fe(3+) in a strong ligand field of low (orthorhombic) symmetry. (31)P NMR experiments demonstrated the presence of bound phosphate in the holo form of nFbp and showed that phosphate can be dialyzed away in the absence of Fe(3+) in apo-nFbp. Finally, an uncorrected Fe(3+/2+) redox potential for Fe-nFbp was determined to be -290 mV (NHE) at pH 6.5, 20 degrees C. Whereas our findings show that nFbp and mammalian transferrin have similar Fe(3+) binding constants and EPR spectra, they differ greatly in their redox potentials. This has implications for the mechanism of Fe transport across the periplasmic space of Gram-negative bacteria.
重组nFbp是一种在致病性奈瑟菌周质空间中发现的铁结合蛋白，其Fe(3+)结合位点已通过物理化学技术进行了表征。在pH 6.5、25℃条件下，存在350微摩尔磷酸盐时，其有效Fe(3+)结合常数测定为2.4×10(18) m(-1)。重组Fe(3+)nFbp的电子顺磁共振光谱给出了g' = 4.3以及在低（正交）对称性强配体场中高自旋Fe(3+)的9个信号特征。(31)P核磁共振实验证明了nFbp全酶形式中存在结合的磷酸盐，并表明在脱辅基nFbp中不存在Fe(3+)时，磷酸盐可通过透析去除。最后，在pH 6.5、20℃条件下，Fe-nFbp未经校正的Fe(3+/2+)氧化还原电位测定为-290 mV（标准氢电极）。尽管我们的研究结果表明nFbp和哺乳动物转铁蛋白具有相似的Fe(3+)结合常数和电子顺磁共振光谱，但它们的氧化还原电位差异很大。这对革兰氏阴性菌周质空间中铁运输的机制具有重要意义。
