Department of Chemistry, Stanford University, 333 Campus Drive Stanford, California 94305, United States.
Department of Biochemistry, The University at Buffalo, 140 Farber Hall, 3435 Main Street, Buffalo, New York 14214, United States.
J Am Chem Soc. 2020 Jun 3;142(22):10087-10101. doi: 10.1021/jacs.0c02384. Epub 2020 May 22.
The multicopper oxidases (MCOs) couple four 1 oxidations of substrate to the 4 reduction of O to HO. These divide into two groups: those that oxidize organic substrates with high turnover frequencies (TOFs) up to 560 s and those that oxidize metal ions with low TOFs, ∼1 s or less. The catalytic mechanism of the organic oxidases has been elucidated, and the high TOF is achieved through rapid intramolecular electron transfer (IET) to the native intermediate (NI), which only slowly decays to the resting form. Here, we uncover the factors that govern the low TOF in Fet3p, a prototypical metallooxidase, in the context of the MCO mechanism. We determine that the NI decays rapidly under optimal turnover conditions, and the mechanism thereby becomes rate-limited by slow IET to the resting enzyme. Development of a catalytic model leads to the important conclusions that proton delivery to the NI controls the mechanism and enables the slow turnover in Fet3p that is functionally significant in Fe metabolism enabling efficient ferroxidase activity while avoiding ROS generation.
多铜氧化酶（MCOs）将底物的四个 1 氧化与 O 到 HO 的四个还原偶联。这些酶分为两类：一类是具有高达 560 s-1 的高周转率（TOF）氧化有机底物的酶，另一类是 TOF 较低、约 1 s 或更短的氧化金属离子的酶。有机氧化酶的催化机制已经阐明，高 TOF 是通过快速的分子内电子转移（IET）到天然中间物（NI）来实现的，NI 只是缓慢地衰减到休眠形式。在这里，我们在 MCO 机制的背景下揭示了控制 Fet3p（一种典型的金属氧化酶）低 TOF 的因素。我们确定，在最佳周转率条件下，NI 迅速衰减，因此该机制受到缓慢 IET 到休眠酶的限制。催化模型的发展得出了重要结论，即向 NI 提供质子控制着机制，并使 Fet3p 中的缓慢周转率成为铁代谢中功能显著的因素，从而能够实现高效的亚铁氧化酶活性，同时避免 ROS 的产生。
