Experimental Evidence for Radical-Polar Crossover in Competition with, Rather Than Preceding, Alkene Formation by...

Ethylene-forming enzyme (EFE) catalyzes a reaction that sets it apart from other iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases. In this reaction, all four oxidizing equivalents of O₂ are unleashed upon 2OG, fragmenting it to ethylene (from C3 and C4) and three fully oxidized C₁ equivalents (from C1, C2, and C5), while the would-be "prime substrate", l-arginine, escapes unmodified. We previously proposed that ethylene formation proceeds by a radical-polar-crossover mechanism involving three unusual steps: (1) formal insertion of O₂ between C1 and C2 of 2OG, forming a succinylperoxycarbonatoiron(II) complex and appending an additional oxygen to C1; (2) radical C-O coupling between a C3-C5-derived propionate-3-yl radical and a C1-derived Fe(III)-coordinated carbonate; and (3) polar fragmentation of the resultant (2-carboxyethyl)carbonatoiron(II) complex to ethylene, CO₂, and carbonate. Here, we used isotopic labeling to distinguish the three C₁ products and stopped-flow infrared (FTIR) spectroscopy to track their formation. The results confirm the prediction that C1 is not directly converted to CO₂, implying that it must indeed become (bi)carbonate. Comparable kinetic data on the A198L variant, which produces ethylene and the abortive product, 3-hydroxypropionate, in similar quantities, reveal that these two products do not, as we had originally proposed, form in competing reactions of a common (2-carboxyethyl)carbonatoiron(II) intermediate. Rather, as suggested by a pair of computational studies separately led by Sayfutyarova and Christov, ethylene is formed in competition with radical coupling by an olefin-forming fragmentation that reduces the Fe(III) cofactor. In other words, crossover to the polar manifold thwarts rather than enables ethylene formation.