Department of Chemistry, Institute of Biochemistry, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, A-1190 Vienna, Austria.
BIMEF Laboratory, Department of Chemistry, University of Antwerp, 2020 Antwerp, Belgium.
Biochemistry. 2021 Mar 2;60(8):621-634. doi: 10.1021/acs.biochem.0c00910. Epub 2021 Feb 15.
Chlorite dismutases (Clds) are heme -containing oxidoreductases that can decompose chlorite to chloride and molecular oxygen. They are divided in two clades that differ in oligomerization, subunit architecture, and the hydrogen-bonding network of the distal catalytic arginine, which is proposed to switch between two conformations during turnover. To understand the impact of the conformational dynamics of this basic amino acid on heme coordination, structure, and catalysis, Cld from sp. PCC7425 was used as a model enzyme. As typical for a clade 2 Cld, its distal arginine 127 is hydrogen-bonded to glutamine 74. The latter has been exchanged with either glutamate (Q74E) to arrest R127 in a salt bridge or valine (Q74V) that mirrors the setting in clade 1 Clds. We present the X-ray crystal structures of Q74V and Q74E and demonstrate the pH-induced changes in the environment and coordination of the heme iron by ultraviolet-visible, circular dichroism, and electron paramagnetic resonance spectroscopies as well as differential scanning calorimetry. The conformational dynamics of R127 is shown to have a significant role in heme coordination during the alkaline transition and in the thermal stability of the heme cavity, whereas its impact on the catalytic efficiency of chlorite degradation is relatively small. The findings are discussed with respect to (i) the flexible loop connecting the N-terminal and C-terminal ferredoxin-like domains, which differs in clade 1 and clade 2 Clds and carries Q74 in clade 2 proteins, and (ii) the proposed role(s) of the arginine in catalysis.
绿泥石双加氧酶（Clds）是一类血红素依赖性氧化还原酶，能够将亚氯酸盐分解为氯离子和分子氧。它们分为两个分支，在寡聚化、亚基结构以及远端催化精氨酸的氢键网络方面存在差异，该精氨酸在周转过程中被提议在两种构象之间切换。为了了解该碱性氨基酸的构象动力学对血红素配位、结构和催化的影响，我们使用 sp. PCC7425 的 Cld 作为模型酶。与典型的 2 型 Cld 一样，其远端精氨酸 127 与谷氨酰胺 74 形成氢键。后者已被谷氨酸（Q74E）取代，以将 R127 固定在盐桥中，或被缬氨酸（Q74V）取代，后者模拟了 1 型 Clds 中的设置。我们呈现了 Q74V 和 Q74E 的 X 射线晶体结构，并通过紫外-可见、圆二色性和电子顺磁共振光谱以及差示扫描量热法证明了 pH 诱导的血红素铁环境和配位变化。结果表明，R127 的构象动力学在碱性转变过程中对血红素配位以及血红素腔的热稳定性具有重要作用，而对亚氯酸盐降解的催化效率的影响相对较小。这些发现与（i）连接 N 端和 C 端铁氧还蛋白样结构域的柔性环有关，该环在 1 型和 2 型 Clds 中存在差异，并且在 2 型蛋白中携带 Q74，以及（ii）精氨酸在催化中的可能作用有关。
