Zhu, GuoliangYan, WupengWang, XinyeCheng, RonghaiNaowarojna, NathcharWang, KunWang, JunSong, HengWang, YuyangLiu, HairongXia, XuekuiCostello, Catherine E.Liu, XuetingZhang, LixinLiu, Pinghua2023-07-142023-07-142022-07-25G. Zhu, W. Yan, X. Wang, R. Cheng, N. Naowarojna, K. Wang, J. Wang, H. Song, Y. Wang, H. Liu, X. Xia, C.E. Costello, X. Liu, L. Zhang, P. Liu. 2022. "Dissecting the Mechanism of the Nonheme Iron Endoperoxidase FtmOx1 Using Substrate Analogues." JACS Au, Volume 2, Issue 7, pp.1686-1698. https://doi.org/10.1021/jacsau.2c002482691-3704https://hdl.handle.net/2144/46453FtmOx1 is a nonheme iron (NHFe) endoperoxidase, catalyzing three disparate reactions, endoperoxidation, alcohol dehydrogenation, and dealkylation, under in vitro conditions; the diversity complicates its mechanistic studies. In this study, we use two substrate analogues to simplify the FtmOx1-catalyzed reaction to either a dealkylation or an alcohol dehydrogenation reaction for structure-function relationship analysis to address two key FtmOx1 mechanistic questions: (1) Y224 flipping in the proposed COX-like model vs α-ketoglutarate (αKG) rotation proposed in the CarC-like mechanistic model and (2) the involvement of a Y224 radical (COX-like model) or a Y68 radical (CarC-like model) in FtmOx1-catalysis. When 13-oxo-fumitremorgin B (7) is used as the substrate, FtmOx1-catalysis changes from the endoperoxidation to a hydroxylation reaction and leads to dealkylation. In addition, consistent with the dealkylation side-reaction in the COX-like model prediction, the X-ray structure of the FtmOx1•CoII•αKG•7 ternary complex reveals a flip of Y224 to an alternative conformation relative to the FtmOx1•FeII•αKG binary complex. Verruculogen (2) was used as a second substrate analogue to study the alcohol dehydrogenation reaction to examine the involvement of the Y224 radical or Y68 radical in FtmOx1-catalysis, and again, the results from the verruculogen reaction are more consistent with the COX-like model.1686-1698Electronic-eCollectionen© 2022 The Authors. Published by American Chemical Society. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0).http://creativecommons.org/licenses/by-nc-nd/4.0/Chemical sciencesArticle2023-02-0810.1021/jacsau.2c002480000-0002-0049-5053 (Song, Heng)0000-0003-1594-5122 (Costello, Catherine E)0000-0002-1322-8253 (Liu, Xueting)0000-0002-9768-559X (Liu, Pinghua)760335