The enzyme soybean lipoxygenase (SLO) has served as a prototype for hydrogen-tunneling reactions, as a result of its unusual kinetic isotope effects (KIEs) and their temperature dependencies. Using a synergy of kinetic, structural, and theoretical studies, we show how the interplay between donor acceptor distance and active-site flexibility leads to catalytic behavior previously predicted by quantum tunneling theory. Modification of the size of two hydrophobic residues by site-specific mutagenesis in SLO reduces the reaction rate 10(4)-fold and is accompanied by an enormous and unprecedented room-temperature KIE. Fitting of the kinetic data to a non-adiabatic model implicates an expansion of the active site that cannot be compensated by donor acceptor distance sampling. A 1.7 angstrom resolution X-ray structure of the double mutant further indicates an unaltered backbone conformation, almost identical side-chain conformations, and a significantly enlarged activesite cavity. These findings show the compelling property of room-temperature hydrogen tunneling within a biological context and demonstrate the very high sensitivity of such tunneling to barrier width.