Institute of Biochemistry, Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, A-1190Vienna, Austria.
BIMEF Laboratory, Department of Chemistry, University of Antwerp, 2000Antwerp, Belgium.
Biochemistry. 2023 Feb 7;62(3):835-850. doi: 10.1021/acs.biochem.2c00696. Epub 2023 Jan 27.
The heme enzyme chlorite dismutase (Cld) catalyzes the degradation of chlorite to chloride and dioxygen. Many questions about the molecular reaction mechanism of this iron protein have remained unanswered, including the electronic nature of the catalytically relevant oxoiron(IV) intermediate and its interaction with the distal, flexible, and catalytically active arginine. Here, we have investigated the dimeric Cld from sp. PCC7425 (Cld) and two variants having the catalytic arginine R127 (i) hydrogen-bonded to glutamine Q74 (wild-type Cld), (ii) arrested in a salt bridge with a glutamate (Q74E), or (iii) being fully flexible (Q74V). Presented stopped-flow spectroscopic studies demonstrate the initial and transient appearance of Compound I in the reaction between Cld and chlorite at pH 5.0 and 7.0 and the dominance of spectral features of an oxoiron(IV) species (418, 528, and 551 nm) during most of the chlorite degradation period at neutral and alkaline pH. Arresting the R127 in a salt bridge delays chlorite decomposition, whereas increased flexibility accelerates the reaction. The dynamics of R127 does not affect the formation of Compound I mediated by hypochlorite but has an influence on Compound I stability, which decreases rapidly with increasing pH. The decrease in activity is accompanied by the formation of protein-based amino acid radicals. Compound I is demonstrated to oxidize iodide, chlorite, and serotonin but not hypochlorite. Serotonin is able to dampen oxidative damage and inactivation of Cld at neutral and alkaline pH. Presented data are discussed with respect to the molecular mechanism of Cld and the pronounced pH dependence of chlorite degradation.
血红素酶亚氯酸盐歧化酶（Cld）催化亚氯酸盐降解为氯化物和氧气。关于这种铁蛋白的分子反应机制，仍有许多问题尚未得到解答，包括催化相关的氧代铁（IV）中间物的电子性质及其与远端、灵活和催化活性的精氨酸的相互作用。在这里，我们研究了来自 sp. PCC7425 的二聚体 Cld 及其两种变体，其中催化精氨酸 R127（i）与谷氨酰胺 Q74 形成氢键（野生型 Cld），（ii）与谷氨酸（Q74E）形成盐桥，或（iii）完全灵活（Q74V）。目前的停流光谱研究表明，在 pH 5.0 和 7.0 下 Cld 与亚氯酸盐之间的反应中，初始和瞬态出现了 Compound I，并且在中性和碱性 pH 下，亚氯酸盐降解的大部分时间内，都存在氧代铁（IV）物种（418、528 和 551nm）的光谱特征占主导地位。将 R127 固定在盐桥中会延迟亚氯酸盐的分解，而增加灵活性会加速反应。R127 的动力学不会影响次氯酸盐介导的 Compound I 的形成，但会影响 Compound I 的稳定性，随着 pH 的增加，其稳定性会迅速下降。活性的降低伴随着蛋白质基氨基酸自由基的形成。证明 Compound I 可以氧化碘化物、亚氯酸盐和 5-羟色胺，但不能氧化次氯酸盐。5-羟色胺能够在中性和碱性 pH 下缓冲 Cld 的氧化损伤和失活。提出的这些数据与 Cld 的分子机制以及亚氯酸盐降解的明显 pH 依赖性有关。
