RESUMO
Important bioactive natural products, including prostaglandin H2 and artemisinin, contain reactive endoperoxides. Known enzymatic pathways for endoperoxide installation require multiple hydrogen-atom transfers (HATs). For example, iron(II)- and 2-oxoglutarate-dependent verruculogen synthase (FtmOx1; EC 1.14.11.38) mediates HAT from aliphatic C21 of fumitremorgin B, capture of O2 by the C21 radical (C21â¢), addition of the peroxyl radical (C21-O-Oâ¢) to olefinic C27, and HAT to the resultant C26â¢. Recent studies proposed conflicting roles for FtmOx1 tyrosine residues, Tyr224 and Tyr68, in the HATs from C21 and to C26â¢. Here, analysis of variant proteins bearing a ring-halogenated tyrosine or (amino)phenylalanine in place of either residue establishes that Tyr68 is the hydrogen donor to C26â¢, while Tyr224 has no essential role. The radicals that accumulate rapidly in FtmOx1 variants bearing a HAT-competent tyrosine analog at position 68 exhibit hypsochromically shifted absorption and, in cases of fluorine substitution, 19F-coupled electron-paramagnetic-resonance (EPR) spectra. By contrast, functional Tyr224-substituted variants generate radicals with unaltered light-absorption and EPR signatures as they produce verruculogen. The alternative major product of the Tyr68Phe variant, which forms competitively with verruculogen also in wild-type FtmOx1 in 2H2O and in the variant with the less readily oxidized 2,3-F2Tyr at position 68, is identified by mass spectrometry and isotopic labeling as the 26-hydroxy-21,27-endoperoxide compound formed after capture of another equivalent of O2 by the longer lived C26â¢. The results highlight the considerable chemical challenges the enzyme must navigate in averting both oxygen rebound and a second O2 coupling to obtain verruculogen selectively over other possible products.