An intriguing but controversial hypothesis has appeared that "The optimization of enzyme catalysis may entail the evolutionary implementation of chemical strategies that increase the probability of tunneling and thereby accelerate the reaction rate" (Kohen, A.; Klinman, J. P. Acc. Chem. Res. 1998, 31, 397). Restated, enzymes may have evolved to enhance quantum mechanical tunneling by coupling to protein low v modes that squeeze the reacting centers together in, for example, their H, atom abstraction reactions. Such a putative "protein squeezing" mechanism would enhance hydrogen quantum mechanical tunneling by reducing the barrier width. An alternative hypothesis is that enzymes do not enhance tunneling, but simply exploit the same amount of tunneling present in their enzyme-free solution reactions, if those reactions occur. A third, conceivable hypothesis is that enzymes might even inadvertently decrease the amount of tunneling as an undesired result of increasing the barrier width while reducing the barrier height. Testing these hypotheses experimentally requires the extremely rare event of being able to measure the amount of tunneling both in the enzyme system and in a very similar if not identical reaction in enzyme-free solution. This has been accomplished experimentally in only one prior case, our recent study of AdoCbI (coenzyme B-12) and 8-Meo-AdoCbI undergoing enzyme-like H, abstraction reactions (Doll, K. M.; Bender, B. R.; Finke, R. G. to J. Am. Chem. Soc. 2003, in press). The data there reveal no change in the level of tunneling within or outside of the enzyme in comparison to the best literature data for an AdoCbl-dependent enzyme, methylmalonyl-CoA mutase. However, that first system suffers from two limitations: the measurement of the KIE (kinetic isotope effect) data in a nonenzymic 80-110 degreesC temperature range; and lower precision data than desired due to the HPLC-MS method required for one of the KIE analyses. These limitations have now been overcome by the synthesis, then thermolysis and KIE study vs temperature of the H-circle abstraction reaction of beta-neopentylcobalamin (beta-NpCbI) in ethylene glycol-do and ethylene glycol-d(4). This is the first experimental test of Klinman's hypothesis using KIE data obtained at enzyme-relevant temperatures. The key data obtained are as follows: deuterium KIEs of 23.1 +/- 3.0 at 40 degreesC to 39.0 +/- 2.3 at 10 degreesC; an activation energy difference E-D - E-H of 3.1 +/- 0.3 kcal mol(-1); and a pre-exponential factor ratio A(H)/A(D) of 0.14 +/- 0.07. Moreover, our now three sets of data (NpCbI; AdoGbI; 8-MeOAdoCbI) are shown to lie on the same In KIE vs 1/T linear plot yielding a set of enzyme-temperature-relevant, high-precision KIE, E-D - E-H, and A(H)/A(D) data over a relatively large, 110 degreesC temperature range. Significantly, the enzyme-free solution KIE, ED - EH, and AH/AD are identical within experimental error to those for methylmalonyl-CoA mutase. This finding leads to the conclusion that there is no enzymic enhancement of the tunneling in at least this B-12-dependent enzyme. This B12 enzyme does, however, exploit the same (unchanged) level of tunneling measured for the nonenzymic, Ad(circle) solution H-circle abstraction reaction. A discussion is presented of the still open question of if this first experimental finding, of "no enzymic enhancement of tunneling" in one B-12-dependent enzymic system, is likely to prove more general or not.