Using α-secondary kinetic isotope effects (2° KIEs) in conjunction with primary (1°) KIEs, we have investigated the mechanism of environmentally coupled hydrogen tunneling in the reductive half-reactions of two homologous flavoenzymes, morphinone reductase (MR) and pentaerythritol tetranitrate reductase (PETNR). We find exalted 2° KIEs (1.17-1.18) for both enzymes, consistent with hydrogen tunneling. These 2° KIEs, unlike 1° KIEs, are independent of promoting motions-a nonequilibrium pre-organization of cofactor and active site residues that is required to bring the reactants into a "tunneling-ready" configuration. That these 2° KIEs are identical suggests the geometries of the "tunneling-ready" configurations in both enzymes are indistinguishable, despite the fact that MR, but not PETNR, has a clearly temperature-dependent 1° KIE. The work emphasizes the benefit of combining studies of 1° and 2° KIEs to report on pre-organization and local geometries within the context of contemporary environmentally coupled frameworks for H-tunneling. © 2006 American Chemical Society.