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• 作者：Sangjin Hong ; Wagner B. de Almeida ; Alexander T. Taguchi ; Rimma I. Samoilova ; Robert B. Gennis ; Patrick J. O鈥橫alley ; Sergei A. Dikanov ; Antony R. Crofts
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：September 30, 2014
• 年：2014
• 卷：53
• 期：38
• 页码：6022-6031
• 全文大小：367K
• ISSN：1520-4995

文摘

Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe ¹³C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with l-methionine with the side chain methyl group ¹³C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the ¹³C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Q_i site of the bc₁ complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of ¹³C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc₁ complex (Protein Data Bank entry 1PP9) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly.