The Stretching Frequencies of Bound Alkyl Isocyanides Indicate Two Distinct Ligand Orientations within the Distal Pocket of Myoglobin
The Stretching Frequencies of Bound Alkyl Isocyanides Indicate Two Distinct Ligand Orientations within the Distal Pocket of Myoglobin
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DOI：
10.1021/bi100172c
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发表时间：
2010-06-22
期刊：
影响因子：
2.9
通讯作者：
Olson, John S.
中科院分区：
文献类型：
--
作者：
Blouin, George C.;Olson, John S.
烷基异氰化物（CNR）与抹香鲸肌红蛋白（Mb）结合后，其红外光谱由中心约为2175 cm（-1）的单一nu（CN）峰变为中心约为2075 cm（-1）和2125 cm（-1）的两个峰。低频和高频峰分别归属于内和外构象。在构象中，配体指向蛋白质内部，远端His 64（E7）处于闭合位置，向结合的异氰基基团提供H-键，增强背键，并降低C-N键顺序。在外构象中，配体侧链通过His 64向外旋转打开的通道指向溶剂。结合位点附近正极性的丧失导致C-N键序的增加。对这一解释的支持有三个方面：（1）当L1 is 64（E7）的I-I-键捐赠发生时，MbCO复合物出现类似的向较低频率的偏移;（2）对于与H64 A或H64 L Mb突变体或与皂胶束中螯合的原血红素结合的CNR，观察到在2125 cm（-1）附近只有一个峰，表明非极性环境;（3）基于FTIR光谱的构象不稳定分数与纳秒激光光解后的成对复合分数密切相关。内烷基侧链构象导致光解离的配体被“卡”在远端口袋中，促进内部重新结合，而外构象抑制成对重组，因为部分配体已经在开放的E7通道中，准备快速逃逸。
The FTIR spectra for alkyl isocyanides (CNRs) change from a single nu(CN) band centered at similar to 2175 cm(-1) to two peaks at similar to 2075 and similar to 2125 cm(-1) upon binding to sperm whale myoglobin (Mb). The low- and high-frequency peaks have been assigned to in and out conformations, respectively. In the in conformation, the ligand is pointing toward the protein interior, and the distal His64(E7) is in a closed position, donates a H-bond to the bound isocyano group, enhances back-bonding, and lowers the C-N bond order. In the out conformation, the ligand side chain points toward solvent through a channel opened by outward rotation of His64. Loss of positive polarity near the binding site causes an increase in C-N bond order. Support for this interpretation is threefold: (1) similar shifts to lower frequency occur for MbCO complexes when I-I-bond donation from L1is64(E7) occurs; (2) only one peak at similar to 2125 cm(-1) indicative of an apolar environment, is observed for CNRs bound to H64A or H64L Mb mutants or to chelated protoheme in soap micelles; and (3) the fraction of in conformation based on FTI R spectra correlates strongly with the fraction of geminate recombination after nanosecond laser photolysis. The in alkyl side chain conformation causes the photodissociated ligand to be "stuck" in the distal pocket, promoting internal rebinding, whereas the out conformation inhibits geminate recombination because part of the ligand is already in an open E7 channel, poised for rapid escape.