Ludwiczek Susanne, Rosell Federico I, Ludwiczek Martin L, Mauk A Grant
Department of Biochemistry and Molecular Biology and the UBC Centre for Blood Research, Life Sciences Centre, 2350 Health Sciences Mall, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Biochemistry. 2008 Jan 15;47(2):753-61. doi: 10.1021/bi701793a. Epub 2007 Dec 20.
Recent studies of mutant mice with compromised ability to absorb dietary iron have identified involvement of two integral membrane proteins in the intestinal epithelial lining in iron uptake, a divalent metal ion transporter and a ferric reductase. The current study concerns the recombinant expression, purification, and initial spectroscopic characterization of a recombinant form of the human ferric reductase that was expressed and purified as the apoprotein from Escherichia coli. Reconstitution of the recombinant protein with ferriprotoporphyrin IX produced a red product with Soret (Fe3+, lambdamax 413.5 nm; Fe2+, lambdamax = 426 nm) and visible absorption maxima indicative of bisimidazole axial coordination. This observation was confirmed by electron paramagnetic resonance and magnetic circular dichroism spectroscopy. Titration of apo-Dcytb with ferriprotoporphyrin IX was consistent with the binding of two heme groups to the protein as predicted by the phylogenetic relationship of this protein to the cytochrome b561 family. Similar titrations and spectroscopic studies of two double variants of Dcytb, each lacking a pair of histidyl residues (H50 and H120 or H86 and H159) proposed on the basis of sequence alignment with other members of the cytochrome b561 family to provide axial ligands to bound heme, indicated that these variants were able to bind just one heme group each.
最近对饮食中铁吸收能力受损的突变小鼠的研究表明，肠上皮衬里中的两种整合膜蛋白参与了铁的摄取，即二价金属离子转运蛋白和铁还原酶。当前的研究涉及一种重组形式的人铁还原酶的重组表达、纯化及初步光谱表征，该重组酶以脱辅基蛋白形式从大肠杆菌中表达并纯化。用亚铁原卟啉IX对重组蛋白进行重构产生了一种红色产物，其Soret吸收峰（Fe3+，λmax = 413.5 nm；Fe2+，λmax = 426 nm）和可见吸收峰表明存在双咪唑轴向配位。电子顺磁共振和磁圆二色光谱证实了这一观察结果。脱辅基Dcytb用亚铁原卟啉IX滴定，结果与两个血红素基团与该蛋白的结合情况一致，这是根据该蛋白与细胞色素b561家族的系统发育关系预测得出的。基于与细胞色素b561家族其他成员的序列比对推测，Dcytb的两个双变体各自缺少一对组氨酸残基（H50和H120或H86和H159），而这些组氨酸残基被认为可为结合的血红素提供轴向配体，对这两个双变体进行的类似滴定和光谱研究表明，这些变体各自只能结合一个血红素基团。
