Sevrioukova Irina F, Garcia Carlos, Li Huiying, Bhaskar B, Poulos Thomas L
Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92612-3900, USA.
J Mol Biol. 2003 Oct 17;333(2):377-92. doi: 10.1016/j.jmb.2003.08.028.
Stability of the [2Fe-2S]-containing putidaredoxin (Pdx), the electron donor to cytochrome P450cam in Pseudomonas putida, was improved by mutating non-ligating cysteine residues, Cys73 and Cys85, to serine singly and in combination. The increasing order of stability is Cys73Ser/Cys85Ser>Cys73Ser>Cys85Ser>WT Pdx. Crystal structures of Cys73Ser/Cys85Ser and Cys73Ser mutants of Pdx, solved by single-wavelength anomalous dispersion phasing using the [2Fe-2S] iron atoms to 1.47 A and 1.65 A resolution, respectively, are nearly identical and very similar to those of bovine adrenodoxin (Adx) and Escherichia coli ferredoxin. However, unlike the Adx structure, no motion between the core and interaction domains of Pdx is observed. This higher conformational stability of Pdx might be due to the presence of a more extensive hydrogen bonding network at the interface between the two structural domains around the conserved His49. In particular, formation of a hydrogen bond between the side-chain of Tyr51 and the carbonyl oxygen atom of Glu77 and the presence of two well-ordered water molecules linking the interaction domain and the C-terminal peptide to the core of the molecule are unique to Pdx. The folding topology of the NMR model is similar to that of the X-ray structure of Pdx. The overall rmsd of Calpha positions between the two models is 1.59 A. The largest positional differences are observed for residues 18-21 and 33-37 in the loop regions and the C terminus. The latter two peptides display conformational heterogeneity in the crystal structures. Owing to flexibility, the aromatic ring of the C-terminal Trp106 can closely approach the side-chains of Asp38 and Thr47 (3.2-3.9 A) or move away and leave the active site solvent-exposed. Therefore, Trp106, previously shown to be important in the Pdr-to-Pdx and Pdx-to-P450cam electron transfer reactions is in a position to regulate and/or mediate electron transfer to or from the [2Fe-2S] center of Pdx.
恶臭假单胞菌中细胞色素P450cam的电子供体、含[2Fe-2S]的恶臭假单胞菌素（Pdx），通过将非配位半胱氨酸残基Cys73和Cys85单独或组合突变为丝氨酸，其稳定性得到了提高。稳定性增加的顺序为：Cys73Ser/Cys85Ser > Cys73Ser > Cys85Ser >野生型Pdx。利用[2Fe-2S]铁原子通过单波长反常色散相位法分别以1.47 Å和1.65 Å的分辨率解析得到的Pdx的Cys73Ser/Cys85Ser和Cys73Ser突变体的晶体结构几乎相同，且与牛肾上腺皮质铁氧还蛋白（Adx）和大肠杆菌铁氧还蛋白的晶体结构非常相似。然而，与Adx结构不同的是，未观察到Pdx的核心结构域与相互作用结构域之间的运动。Pdx这种更高的构象稳定性可能是由于在保守的His49周围两个结构域之间的界面处存在更广泛的氢键网络。特别是，Tyr51的侧链与Glu77的羰基氧原子之间形成氢键，以及存在两个排列有序的水分子将相互作用结构域和C端肽与分子核心相连，这些都是Pdx所特有的。NMR模型的折叠拓扑结构与Pdx的X射线结构相似。两个模型之间Cα位置的总体均方根偏差为1.59 Å。在环区和C端的18 - 21位和33 - 37位残基观察到最大的位置差异。后两个肽段在晶体结构中表现出构象异质性。由于具有灵活性，C端Trp106的芳香环可以紧密靠近Asp38和Thr47的侧链（3.2 - 3.9 Å），或者移开并使活性位点暴露于溶剂中。因此，先前已证明在Pdr到Pdx以及Pdx到P450cam电子转移反应中起重要作用的Trp106，能够调节和/或介导电子向Pdx的[2Fe-2S]中心或从该中心转移。
