Experimental and Computational Investigations of Oxygen Reactivity in a Heme and Tyrosyl Radical-containing Fatty Acid α-(Di)oxygenase

Huff, G. S.; Doncheva, I. S.; Brinkley, D. W.; Angeles-Boza, A. M.; Mukherjee, A.; Cramer, C. J.; Roth, J. P.
Biochemistry 2011, 50, 7375 (doi:10.1021/bi201016h).

Rice α-(di)oxygenase mediates the regio and stereo-specific oxidation of fatty acids using a persistent catalytic tyrosyl radical. Experiments conducted in the physiological O₂ concentration range, where initial hydrogen atom abstraction from the fatty acid occurs in a kinetically reversible manner, are described. Our findings indicate that O₂-trapping of an α-carbon radical is likely to reversibly precede reduction of a 2-(R)-peroxyl radical intermediate in the first irreversible step. A mechanism of concerted proton-coupled electron transfer is proposed on the basis of natural abundance oxygen-18 kinetic isotope effects, deuterium kinetic isotope effects and calculations at the density functional level of theory, which predict a polarized transition state in which electron transfer is advanced to a greater extent than proton transfer. The approach outlined should be useful for identifying mechanisms of concerted proton-coupled electron transfer in a variety of oxygen-utilizing enzymes.