Holzbaur I E, English A M, Ismail A A
Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada.
Biochemistry. 1996 Apr 30;35(17):5488-94. doi: 10.1021/bi952233m.
Fourier transform infrared (FTIR) spectroscopy was employed to examine the thermal denaturation of the Fe(III), Fe(II), and Fe(II)-CO forms of cytochrome c peroxidase and horseradish peroxidase in phosphate buffer at pD 7.0. The amide I' regions of the deconvolved spectra are consistent with predominantly alpha-helical secondary structure around room temperature, but the alpha-helical absorption of the two peroxidases differs significantly; bands assigned to alpha-helical components occur at 1659 and 1649 cm-1 in horseradish peroxidase and at 1652 and 1637 cm-1 in cytochrome c peroxidase. The thermal denaturation mechanisms of the peroxidases also vary. All three forms of cytochrome c peroxidase retain their secondary structure up to 50 degrees C, when bands characteristic of aggregation (1616 and 1684 cm-1) appear in the amide I' region, and above 55 degrees C rapid loss of secondary structure is accompanied by enhanced aggregation. In horseradish peroxidase, on the other hand, the Fe(III) and Fe(II) states exhibit dissimilar denaturation mechanisms. Slow, gradual alteration of secondary structure is observed for Fe(III) horseradish peroxidase on heating, and polypeptide unfolding appears to be complete around 90 degrees C, without aggregation. In Fe(II) and Fe(II)-CO horseradish peroxidase, aggregation bands appear at approximately 55 degrees C, signaling the onset of denaturation. Frequency shifts in the v(CO) bands above room temperature reveal the conformational changes in the heme cavity precede global conformational changes in cytochrome c peroxidase but not in horseradish peroxidase. The reduction in amide II intensities, due to peptide H-D exchange on heating the peroxidases in D2O, indicates the formation above room temperature of partially unfolded states with increased solvent accessibility but intact secondary structures.
利用傅里叶变换红外（FTIR）光谱研究了细胞色素c过氧化物酶和辣根过氧化物酶的Fe(III)、Fe(II)及Fe(II)-CO形式在pD 7.0的磷酸盐缓冲液中的热变性。解卷积光谱的酰胺I'区域在室温附近主要与α-螺旋二级结构一致，但两种过氧化物酶的α-螺旋吸收有显著差异；在辣根过氧化物酶中，归属于α-螺旋成分的谱带出现在1659和1649 cm-1处，而在细胞色素c过氧化物酶中则出现在1652和1637 cm-1处。过氧化物酶的热变性机制也有所不同。细胞色素c过氧化物酶的所有三种形式在高达50℃时都保留其二级结构，此时酰胺I'区域出现聚集特征谱带（1616和1684 cm-1），而在55℃以上，二级结构迅速丧失并伴有聚集增强。另一方面，在辣根过氧化物酶中，Fe(III)和Fe(II)状态表现出不同的变性机制。加热时，Fe(III)辣根过氧化物酶的二级结构发生缓慢、逐渐的变化，多肽解折叠在约90℃时似乎完成，且无聚集现象。在Fe(II)和Fe(II)-CO辣根过氧化物酶中，聚集谱带出现在约55℃，表明变性开始。室温以上v(CO)谱带的频率变化表明，细胞色素c过氧化物酶血红素腔中的构象变化先于整体构象变化，而辣根过氧化物酶则不然。在D2O中加热过氧化物酶时，由于肽H-D交换导致酰胺II强度降低，这表明在室温以上形成了部分解折叠状态，溶剂可及性增加，但二级结构完整。
