National Academy of Sciences, Proceedings of the National Academy of Sciences, 26(106), p. 10603-10608, 2009
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Dioxygen (O 2 ) and other gas molecules have a fundamental role in a variety of enzymatic reactions. However, it is only poorly understood which O 2 uptake mechanism enzymes employ to promote efficient catalysis and how general this is. We investigated O 2 diffusion pathways into monooxygenase and oxidase flavoenzymes, using an integrated computational and experimental approach. Enhanced-statistics molecular dynamics simulations reveal spontaneous protein-guided O 2 diffusion from the bulk solvent to preorganized protein cavities. The predicted protein-guided diffusion paths and the importance of key cavity residues for oxygen diffusion were verified by combining site-directed mutagenesis, rapid kinetics experiments, and high-resolution X-ray structures. This study indicates that monooxygenase and oxidase flavoenzymes employ multiple funnel-shaped diffusion pathways to absorb O 2 from the solvent and direct it to the reacting C4a atom of the flavin cofactor. The difference in O 2 reactivity among dehydrogenases, monooxygenases, and oxidases ultimately resides in the fine modulation of the local environment embedding the reactive locus of the flavin.