Mo H, Pochapsky S S, Pochapsky T C
Department of Chemistry, Brandeis University, Waltham, Massachusetts 02454, USA.
Biochemistry. 1999 Apr 27;38(17):5666-75. doi: 10.1021/bi983063r.
Terpredoxin (Tdx) is a 105-residue bacterial ferredoxin consisting of a single polypeptide chain and a single Fe2S2 prosthetic group. Tdx was first identified in a strain of Pseudomonas sp. capable of using alpha-terpineol as sole carbon source. The Tdx gene, previously cloned from the plasmid-encoded terp operon, that carries genes encoding for proteins involved in terpineol catabolism, has been subcloned and expressed as the holoprotein in E. coli. Physical characterization of the expressed Tdx has been performed, and a model for the solution structure of oxidized Tdx (Tdxo) has been determined. High-resolution homo- and heteronuclear NMR data have been used for structure determination in diamagnetic regions of the protein. The structure of the metal binding site (which cannot be determined directly by NMR methods due to paramagnetic broadening of resonances) was modeled using restraints obtained from a crystal structure of the homologous ferredoxin adrenodoxin (Adx) and loose restraints determined from paramagnetic broadening patterns in NMR spectra. Essentially complete 1H and 15N NMR resonance assignments have been made for the diamagnetic region of Tdxo (ca. 80% of the protein). A large five-stranded beta-sheet and a smaller two-stranded beta-sheet were identified, along with three alpha-helices. A high degree of structural homology was observed between Tdx and two other ferredoxins with sequence and functional homology to Tdx for which structures have been determined, Adx and putidaredoxin (Pdx), a homologous Pseudomonas protein. 1H/2H exchange rates for Tdx backbone NH groups were measured for both oxidation states and are rationalized in the context of the Tdx structure. In particular, an argument is made for the importance of the residue following the third ligand of the metal cluster (Arg49 in Tdx, His49 in Pdx, His56 in Adx) in modulating protein dynamics as a function of oxidation state. Some differences between Tdx and Pdx are detected by UV-visible spectroscopy, and structural differences at the C-terminal region were also observed. Tdx exhibits only 2% of the activity of Pdx in turnover assays performed using the reconstituted camphor hydroxylase system of which Pdx is the natural component.
特普雷多辛（Tdx）是一种由105个氨基酸残基组成的细菌铁氧化还原蛋白，由一条多肽链和一个Fe2S2辅基组成。Tdx最初是在一株能够利用α-松油醇作为唯一碳源的假单胞菌属菌株中鉴定出来的。Tdx基因先前是从质粒编码的萜类操纵子中克隆出来的，该操纵子携带编码参与松油醇分解代谢的蛋白质的基因，已被亚克隆并在大肠杆菌中表达为全蛋白。已对表达的Tdx进行了物理表征，并确定了氧化型Tdx（Tdxo）溶液结构的模型。高分辨率的同核和异核NMR数据已用于蛋白质抗磁性区域的结构测定。金属结合位点的结构（由于共振的顺磁加宽而无法通过NMR方法直接确定）是使用从同源铁氧化还原蛋白肾上腺铁氧化还原蛋白（Adx）的晶体结构获得的限制条件以及从NMR光谱中的顺磁加宽模式确定的宽松限制条件进行建模的。基本上已完成了对Tdxo抗磁性区域（约占蛋白质的80%）的1H和15N NMR共振归属。鉴定出一个大的五链β-折叠和一个较小的双链β-折叠，以及三个α-螺旋。在Tdx与另外两种与Tdx具有序列和功能同源性且结构已确定的铁氧化还原蛋白之间观察到高度的结构同源性，即Adx和恶臭假单胞菌铁氧化还原蛋白（Pdx），一种同源的假单胞菌蛋白。测定了Tdx主链NH基团在两种氧化态下的1H/2H交换率，并根据Tdx结构进行了合理化解释。特别是，有人认为金属簇第三个配体之后的残基（Tdx中的Arg49；Pdx中的His49；Adx中的His56）在调节蛋白质动力学作为氧化态的函数方面很重要。通过紫外可见光谱检测到Tdx和Pdx之间的一些差异，并且在C末端区域也观察到结构差异。在使用Pdx作为天然成分的重组樟脑羟化酶系统进行的周转测定中，Tdx的活性仅为Pdx的2%。
