Geeraerts Zachary, Stiller Olivia R, Lukat-Rodgers Gudrun S, Rodgers Kenton R
Department of Chemistry and Biochemistry, North Dakota State University, Fargo, ND 58108.
ACS Catal. 2022 Jul 15;12(14):8641-8657. doi: 10.1021/acscatal.2c01428. Epub 2022 Jul 6.
The heme-based chlorite dismutases catalyze the unimolecular decomposition of chlorite (ClO ) to yield Cl and O. The work presented here shows that chlorite dismutase from (Cld) also catalyzes the decomposition of bromite (BrO ) with the evolution of O ( = (2.0±0.2)×10 s; / = (1.2±0.2)×10 M s at pH 5.2). Stopped-flow studies of this BrO decomposition as a function of pH show that 1) the two-electron oxidized heme, compound I (Cpd I), is the primary accumulating heme intermediate during O evolution in acidic solution, 2) Cpd I and its one-electron reduction product, compound II (Cpd II) are present in varying ratios at intermediate pHs, and 3) only Cpd II is observed at pH 9.0. The pH dependences of Cpd I and Cpd II populations both yield a p of 6.7±0.1 in good agreement with the p of Cld activity with ClO . The observation of a protein-based amino acid radical (AA•) whose appearance coincides with that of Cpd II supports the hypothesis that conversion of Cpd I to Cpd II occurs via proton-coupled electron transfer (PCET) from a heme-pocket amino acid to the oxidized porphyrinate of Cpd I to yield a dead-end decoupled state in which the holes decay at different rates. The site of the amino acid radical is tentatively assigned to Y118, which serves as a H-bond donor to propionate 6 (P6). The favoring of Cpd II:AA• accumulation in alkaline solution is consistent with the amino acid oxidation being rate limited by transfer of its proton to P6 having p 6.7. Examination of reaction mixtures comprising Cld and ClO by resonance Raman and electron paramagnetic resonance spectroscopy reveal formation of Cpd II and •ClO, which forms in preference to the analogous to AA• in the BrO reaction. Addition of ClO to Cpd II did not yield O. Together these results are consistent with heterolytic cleavage of the O-BrO and O-ClO bonds yielding Cpd I, which is the catalytically active intermediate. The long-lived Cpd II that forms subsequently, is inactive toward O production, and diminishes the amount of enzyme available to cycle through the active Cpd I intermediate.
基于血红素的亚氯酸盐歧化酶催化亚氯酸盐（ClO₂⁻）的单分子分解，生成Cl⁻和O₂。本文展示的研究表明，来自[具体来源未提及]的亚氯酸盐歧化酶（Cld）也能催化溴酸盐（BrO₂⁻）的分解并释放O₂（在pH 5.2时，k = (2.0±0.2)×10⁻³ s⁻¹；kcat/Km = (1.2±0.2)×10⁵ M⁻¹ s⁻¹）。对该BrO₂⁻分解随pH变化的停流研究表明：1）两电子氧化的血红素，即化合物I（Cpd I），是酸性溶液中O₂释放过程中主要积累的血红素中间体；2）在中等pH值下，Cpd I及其单电子还原产物化合物II（Cpd II）以不同比例存在；3）在pH 9.0时仅观察到Cpd II。Cpd I和Cpd II含量的pH依赖性均产生pKa为6.7±0.1，这与Cld对ClO₂⁻的活性pKa非常吻合。观察到一种基于蛋白质的氨基酸自由基（AA•），其出现与Cpd II的出现同时发生，这支持了以下假设：Cpd I向Cpd II的转化是通过从血红素口袋氨基酸到Cpd I氧化卟啉的质子耦合电子转移（PCET）发生的，从而产生一种死端解耦状态，其中空穴以不同速率衰减。氨基酸自由基的位点初步确定为Y118，它作为丙酸6（P6）的氢键供体。在碱性溶液中Cpd II:AA•积累的偏好与氨基酸氧化受其质子转移到pKa为6.7的P6的速率限制一致。通过共振拉曼光谱和电子顺磁共振光谱对包含Cld和ClO₂⁻的反应混合物进行检查，揭示了Cpd II和•ClO的形成，在BrO₂⁻反应中•ClO比类似的AA•更易形成。向Cpd II中添加ClO₂⁻不会产生O₂。这些结果共同表明，O - BrO键和O - ClO键的异裂产生了Cpd I，它是催化活性中间体。随后形成的长寿命Cpd II对O₂生成无活性，并减少了可通过活性Cpd I中间体循环的酶的量。
