Spectroscopic characterization of mutations at the Phe41 position in the distal haem pocket of horseradish peroxidase C: structural and functional consequences.
Spectroscopic characterization of mutations at the Phe41 position in the distal haem pocket of horseradish peroxidase C: structural and functional consequences.
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辣根过氧化物酶 C 远端血红袋中 Phe41 位置突变的光谱表征：结构和功能后果。
DOI：
10.1042/bj3630571
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
G. Smulevich
中科院分区：
文献类型：
--
作者：
H. Heering;Andrew T. Smith;G. Smulevich
构建了3个辣根过氧化物酶同工酶C(HRPC)突变体，其中保守的远端芳香族残基Phe(41)被Trp、Val或Ala取代，并与野生型蛋白的性质进行了比较。用共振拉曼光谱、电子吸收光谱和傅里叶变换红外光谱研究了铁和亚铁的状态，以及它们各自的氟化物和一氧化碳络合物，作为远端血袋氢键网络完整性的探针。突变体的催化性能也受到影响，最明显的是HRPC突变体Phe(41)--&Gt；Trp(F41W)。结构模拟表明，F41W突变体的庞大吲哚基团阻断了远端空洞，抑制了氟和CO与血红素铁的结合，严重阻碍了酶与H(2)O(2)形成化合物I的反应。用较小的侧链残基Val或Ala取代突变体导致突变体对芳香族供体苯异羟肟酸(BHA)的亲和力比野生型提高2倍，而空间受阻的F41W突变体根本不能与BHA结合。所有研究的突变都增加了铁六配位高自旋物种的数量。另一方面，突变体的Fe-Im伸展频率与野生型蛋白的相似性表明，远端的血袋突变不会在血球的近端引起任何实质性的变化。HRPC突变体Phe(41)->；Ala-CO和HRPC突变体Phe(41)->；Val-CO的光谱强烈表明，由于血红素相对于螺旋B的二次重排，CO与Arg(38)之间的相互作用减弱。这些HRP突变体的效果与最近注意到的类似的鸡腿菇过氧化物酶(CIP)突变体，特别是Trp突变体有所不同。这些差异可以部分地归因于HRP远端腔比CIP小。
Three mutants of horseradish peroxidase isoenzyme C (HRPC) have been constructed in which the conserved distal aromatic residue Phe(41) has been substituted by Trp, Val or Ala and the properties of the mutant proteins have been compared with that of the wild-type. The ferric and ferrous states have been studied by resonance Raman, electronic absorption and Fourier-transform infrared spectroscopies, together with their respective fluoride and CO complexes as probes for the integrity of the distal haem-pocket hydrogen-bonding network. The catalytic properties of the mutants, most notably the HRPC-mutant Phe(41)-->Trp (F41W) variant, were also affected. Structural modelling suggests that the bulky indole group of the F41W mutant blocks the distal cavity, inhibiting the binding of fluoride and CO to the haem iron, severely impairing the reaction of the enzyme with H(2)O(2) to form Compound I. Substitution with the smaller side-chain residues Val or Ala resulted in a 2-fold increase in the affinity of the mutants for the aromatic donor benzhydroxamic acid (BHA) compared with the wild-type, whereas the sterically hindered F41W mutant was not able to bind BHA at all. All the mutations studied increased the amount of a ferric six-coordinate aquo-high-spin species. On the other hand, the similarity in the Fe-Im stretching frequencies of the mutants and wild-type protein suggests that the distal haem-pocket mutations do not cause any substantive changes on the proximal side of the haem. Spectra of the HRPC mutant Phe(41)-->Ala-CO and the HRPC mutant Phe(41)-->Val-CO complexes strongly suggested a weakening of the interaction between CO and Arg(38) due to a secondary rearrangement of the haem relative to helix B. The effects observed for these HRP mutants were somewhat different from those noted recently for the analogous Coprinus cinereus peroxidase (CIP) mutants, particularly the Trp mutant. These differences can be reconciled in part as being due to the smaller size of the distal cavity of HRP compared with that of CIP.
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影响因子：
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