The hydrogen-atom transfer in soybean lipoxygenase-1 (SLO) exhibits a large kinetic isotope effect on k_cat (KIE = 81) near room temperature and a very weak temperature dependence (E_act = 2.1 kcal/mol). These properties are consistent with H· transfer that occurs entirely by a tunneling event. Mutants of SLO were prepared, and the temperature dependence of the KIE was measured, to test for alterations in the tunneling behavior. All mutants studied exhibit KIEs of similar, large magnitude at 30 °C, despite an up to 3 orders of magnitude change in k_cat. E_act for two of the mutants (Leu⁷⁵⁴ → Ala, Leu⁵⁴⁶ → Ala) is larger than for wild-type (WT), and the KIE becomes slightly more temperature dependent. In contrast, Ile⁵⁵³ → Ala exhibits k_cat and E_act parameters similar to wild-type soybean lipoxygenase-1 (WT-SLO) for protiated substrate; however, the KIE is markedly temperature dependent. The behavior of the …