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首页> 外文期刊>Biochemistry >The Roles of Tyr(CD1)and Trp(G8)in Mycobacterium tuberculosis Truncated Hemoglobin O in Ligand Binding and on the Heme Distal Site Architecture
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The Roles of Tyr(CD1)and Trp(G8)in Mycobacterium tuberculosis Truncated Hemoglobin O in Ligand Binding and on the Heme Distal Site Architecture

机译：Tyr（CD1）和Trp（G8）在结核分枝杆菌截断的血红蛋白O在配体结合和血红素远端位点结构中的作用

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The crystal structure of the cyano-met form of Mt-trHbO revealed two unusual distal residues Y(CD1)and W(G8)forming a hydrogen-bond network with the heme-bound ligand[Milani,M.,et al.(2003)Proc.Natl.Acad.Sci.U.S.A.100,5766-5771].W(G8)is an invariant residue in group II and group III trHbs and has no counterpart in other globins.A previous study reported that changing Y(CD1)for a Phe causes a significant increase in the O2 combination rate,but almost no change in the O2 dissociation rate[Ouellet,H.,et al.(2003)Biochemistry 42,5764-5774].Here we investigated the role of the W(G8)in ligand binding by using resonance Raman spectroscopy,stopped-flow spectrophotometry,and X-ray crystallography.For this purpose,W(G8)was changed,by site-directed mutagenesis,to a Phe in both the wild-type protein and the mutant Y(CD1)F to create the single mutant W(G8)F and the double mutant Y(CD1)F/W(G8)F,respectively.Resonance Raman results suggest that W(G8)interacts with the heme-bound O2 and CO,as evidenced by the increase of the Fe-O2 stretching mode from 559 to 564 cm~(-1)and by the lower frequency of the Fe-CO stretching modes(514 and 497 cm~(-1))compared to that of the wild-type protein.Mutation of W(G8)to Phe indicates that this residue controls ligand binding,as evidenced by a dramatic increase of the combination rates of both O2 and CO.Also,the rate of O2 dissociation showed a 90-1000-fold increase in the W(G8)F and Y(CD1)F/W(G8)F mutants,that is in sharp contrast with the values obtained for the other distal mutants Y(B10)F and Y(CD1)F[Ouellet,H.,et al.(2003)Biochemistry 42,5764-5774].Taken together,these data indicate a pivotal role for the W(G8)residue in O2 binding and stabilization.
机译：Mt-trHbO氰基形式的晶体结构揭示了两个异常的远端残基Y（CD1）和W（G8）与血红素结合的配体形成氢键网络[Milani，M.，et al。（2003） Natl.Acad.Sci.USA100,5766-5771]。W（G8）是第II组和第III组trHbs中的不变残基，在其他珠蛋白中没有对应物。先前的研究报道，改变Y（CD1） Phe引起O2结合率显着增加，但O2解离率几乎没有变化[Ouellet，H.，et al。（2003）Biochemistry 42,5764-5774]。在这里我们研究了W的作用（G8）通过共振拉曼光谱，停止流式分光光度法和X射线晶体学分析与配体结合。为此，通过定点诱变将W（G8）转变为两种野生型蛋白中的Phe突变体Y（CD1）F分别形成单个突变体W（G8）F和双突变体Y（CD1）F / W（G8）F。共振拉曼实验表明W（G8）与血红素相互作用。结合氧气和一氧化碳，作为证据d是由于Fe-O2拉伸模式从559 cm〜（-1）增加到564 cm〜（-1），Fe-CO拉伸模式的频率（514和497 cm〜（-1））相对于野生模式较低W（G8）与Phe的突变表明该残基控制配体结合，O2和CO的结合率显着增加证明了这一点。此外，O2的解离速率显示90-1000倍W（G8）F和Y（CD1）F / W（G8）F突变体的增加，与其他远端突变体Y（B10）F和Y（CD1）F获得的值形成鲜明对比。 H.，et al。（2003）Biochemistry 42,5764-5774]。这些数据表明，W（G8）残基在O 2结合和稳定中起着关键作用。

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