Ugarova N N, Savitski A P, Berezin I V
Biochim Biophys Acta. 1981 Dec 15;662(2):210-9. doi: 10.1016/0005-2744(81)90032-2.
Similarity of the protein tertiary structures of the native horseradish peroxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) and protoporphyrin-apoperoxidase complex has been shown on the basis of identity of the tryptophan fluorescence parameter at pH 2.0-8.0 and of the circular dichroism spectra of the two proteins. Absorption and fluorescence spectra have been obtained for protoporphyrin in the complex in the pH range 7.0-1.6. A shift in the apparent pK by 4 units has been observed for protonation of the protoporphyrin pyrrolic ring in the complex. From this shift, the dielectric constant has been evaluated for the heme pocket of the peroxidase (approx. 20). Fluorescence quantum yield of protoporphyrin in the complex increased with pH decreasing from 5.0 to 3.5, whereas the spectrum pattern and fluorescence lifetime did not change. The ions, I- and [Fe(CN)6]-4, peroxidase substrates, did not quench the protoporphyrin fluorescence in the complex at about neutral pH, whereas the quenching markedly enhanced with lowering pH. The bimolecular constant for the I- -quenching of the porphyrin fluorescence on the complex showed a pH-dependence similar to that of the bimolecular rate constant for the reaction of peroxidase compound I with I-. Mechanism for I- oxidation at an acid pH in the presence of peroxidase has been proposed.
基于在pH 2.0 - 8.0时色氨酸荧光参数以及两种蛋白质的圆二色光谱的一致性，已证明天然辣根过氧化物酶（供体：过氧化氢氧化还原酶，EC 1.11.1.7）与原卟啉 - 脱辅基过氧化物酶复合物的蛋白质三级结构具有相似性。已获得复合物中原卟啉在pH范围7.0 - 1.6内的吸收光谱和荧光光谱。观察到复合物中原卟啉吡咯环质子化时表观pK有4个单位的偏移。据此偏移，评估了过氧化物酶血红素口袋的介电常数（约为20）。复合物中原卟啉的荧光量子产率随pH从5.0降至3.5而增加，而光谱模式和荧光寿命未改变。过氧化物酶底物I⁻和[Fe(CN)₆]⁴⁻在接近中性pH时不会淬灭复合物中原卟啉的荧光，而随着pH降低淬灭作用明显增强。复合物上卟啉荧光被I⁻淬灭的双分子常数显示出与过氧化物酶化合物I与I⁻反应的双分子速率常数相似的pH依赖性。已提出在过氧化物酶存在下酸性pH时I⁻氧化的机制。
