Dynamic structural fluctuations occurring over a broad range of timescales are now known to facilitate the catalytic function of enzymes, but there is less comprehensive experimental evidence linking fast-timescale, high frequency motions to the reaction coordinate. Interest in the role of such motions has recently surged and been the subject of intensive experimental efforts, in part due to the identification of enzymatic hydride tunnelling reactions. This mechanism involves transiently degenerate product and reactant states, which enable H-transfer to occur instantaneously without the need to surmount the activation barrier associated with traditional transition-state based models of enzyme catalysis. The primary gauge of tunnelling in enzyme-catalysed reactions is the identification of temperature dependent kinetic isotope effects (KIEs), i.e. the relative rates of a reaction where the transferred atom is substituted for an alternate isotope. The identification of temperature-, and also pressure-, dependent KIEs has resulted in the emergence of new models of describing enzymatic H-transfer. These invoke a role for fast-timescale protein motions that 'promote' transfer via tunnelling. A popular model system for studying enzymatic H-tunnelling reactions is Pentaerythritol tetranitrate reductase, which belongs to the Old Yellow Enzyme (OYE) family of ene-reductases. These nicotinamide coenzyme dependent oxidoreductases catalyse the stereospecific reduction of alpha/β-unsaturated alkene containing substrates. Here, the importance of donor-acceptor distances in determining the observed rate of PETNR reduction with NAD(P)H is probed via a detailed structural and kinetic analysis of site-directed variants. In addition, an investigation of distance-dependent Nuclear Overhauser effects via Nuclear Magnetic Resonance (NMR) spectroscopy is undertaken to assess active site organisation and measure donor-acceptor distances in PETNR-substrate complexes. A variable pressure NMR study reveals how NOE build- up is perturbed in high-energy conformers favoured as a result of the application of increased hydrostatic pressures. Recently there has been interest in exploiting the stereoselective properties of reactions catalysed by ene-reductase enzymes for use in biocatalytic reactions to produce industrially valuable compounds from renewable sources. The reactions of PETNR and additional OYE enzymes, Thermophilic old yellow enzyme and Xenobiotic reductase A, with both natural coenzymes and a set of synthetic Nicotinamide Coenzyme Biomimetics (NCBs) are also characterised. The NCBs represent affordable and fast-reacting alternatives to the physiological coenzymes. Reactions with NCBS are also shown to proceed via a tunnelling mechanism and furthermore, that enhanced donor-acceptor sampling correlates with the faster reactivity seen with these compounds.