Abstract

Chlorite dismutase is a heme enzyme that catalyzes the conversion of the toxic compound ClO₂⁻ (chlorite) to innocuous Cl⁻ and O₂. The reaction is a very rare case of enzymatic O–O bond formation, which has sparked the interest to elucidate the reaction mechanism using pre-steady-state kinetics. During stopped-flow experiments, spectroscopic and structural changes of the enzyme were observed in the absence of a substrate in the time range from milliseconds to minutes. These effects are a consequence of illumination with UV–visible light during the stopped-flow experiment. The changes in the UV–visible spectrum in the initial 200 s of the reaction indicate a possible involvement of a ferric superoxide/ferrous oxo or ferric hydroxide intermediate during the photochemical inactivation. Observed EPR spectral changes after 30 min reaction time indicate the loss of the heme and release of iron during the process. During prolonged illumination, the oligomeric state of the enzyme changes from homo-pentameric to monomeric with subsequent protein precipitation. Understanding the effects of UV–visible light illumination induced changes of chlorite dismutase will help us to understand the nature and mechanism of photosensitivity of heme enzymes in general. Furthermore, previously reported stopped-flow data of chlorite dismutase and potentially other heme enzymes will need to be re-evaluated in the context of the photosensitivity.

Graphic abstract

Illumination of recombinantly expressed Azospira oryzae Chlorite dismutase (AoCld) with a high-intensity light source, common in stopped-flow equipment, results in disruption of the bond between Fe^III and the axial histidine. This leads to the enzyme losing its heme cofactor and changing its oligomeric state as shown by spectroscopic changes and loss of activity.

中文翻译：

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特征明确的血红素酶亚氯酸盐歧化酶的意外光敏性

摘要

亚氯酸盐氧化物歧化酶是一种催化有毒化合物CLO的转化血红素酶₂ ^- （亚氯酸盐）成无害氯^-和O ₂。该反应是酶O-O键形成的非常罕见的情况，这激发了人们利用稳态前动力学阐明反应机理的兴趣。在停止流实验期间，在毫秒到分钟的时间范围内，在没有底物的情况下观察到酶的光谱和结构变化。这些影响是在停止流实验期间用紫外线可见光照明的结果。在反应开始的200 s内，紫外-可见光谱的变化表明，在光化学灭活过程中，可能会涉及到超铁/氧亚铁或氢氧化铁中间体。30分钟反应时间后观察到的EPR光谱变化表明在此过程中血红素的损失和铁的释放。在长时间照明下 酶的低聚状态从同五聚体变为单体，随后发生蛋白质沉淀。了解紫外线可见光照射引起的亚氯酸盐歧化酶变化的作用，将有助于我们大致了解血红素酶的光敏性质和机理。此外，在光敏性的背景下，需要重新评估先前报道的亚氯酸盐歧化酶和其他可能的血红素酶的停止流量数据。

图形摘要

在停止流设备中常见的高强度光源对重组表达的米曲霉亚氯螺旋藻歧化酶（Ao Cld）的照明导致Fe ^III与轴向组氨酸之间的键断裂。如光谱变化和活性丧失所示，这导致酶失去其血红素辅因子并改变其低聚状态。