We present an atomic-level description of the reaction chemistry of an enzyme-catalyzed reaction dominated by proton tunneling. By solving structures of reaction intermediates at near-atomic resolution, we have identified the reaction pathway for tryptamine oxidation by aromatic amine dehydrogenase. Combining experiment and computer simulation, we show proton transfer occurs predominantly to oxygen O2 of Asp(128)beta in a reaction dominated by tunneling over approximately 0.6 angstroms. The role of long-range coupled motions in promoting tunneling is controversial. We show that, in this enzyme system, tunneling is promoted by a short-range motion modulating proton-acceptor distance and no long-range coupled motion is required.

Enzymatic breakage of the substrate C-H bond by Methylophilus methyltrophus (sp. W3A1) methylamine dehydrogenase (MADH) has been studied by stopped-flow spectroscopy. The rate of reduction of the tryptophan tryptophylquinone (TTQ) cofactor has a large kinetic isotope effect (KIE = 16.8 +/- 0.5), and the KIE is independent of temperature. Analysis of the temperature dependence of C-H bond breakage revealed that extreme (ground state) quantum tunneling is responsible for the transfer of the hydrogen nucleus. Reaction rates are strongly dependent on temperature, indicating thermally induced, vibrational motion drives the H-transfer reaction. The data provide direct experimental evidence for enzymatic bond breakage by extreme tunneling driven by vibrational motion of the protein scaffold. The results demonstrate that classical transition state theory and its tunneling derivatives do not adequately describe this enzymatic reaction.

Here we report the crystal structures of a ternary electron transfer complex showing extensive motion at the protein interface. This physiological complex comprises the iron-sulfur flavoprotein trimethylamine dehydrogenase and electron transferring flavoprotein (ETF) from Methylophilus methylotrophus. In addition, we report the crystal structure of free ETF. In the complex, electron density for the FAD domain of ETF is absent, indicating high mobility. Positions for the FAD domain are revealed by molecular dynamics simulation, consistent with crystal structures and kinetic data. A dual interaction of ETF with trimethylamine dehydrogenase provides for dynamical motion at the protein interface: one site acts as an anchor, thereby allowing the other site to sample a large range of interactions, some compatible with rapid electron transfer. This study establishes the role of conformational sampling in multi-domain redox systems, providing insight into electron transfer between ETFs and structurally distinct redox partners.

The mechanism of flavin reduction in morphinone reductase (MR) and pentaerythritol tetranitrate (PETN) reductase, and flavin oxidation in MR, has been studied by stopped-flow and steady-state kinetic methods. The temperature dependence of the primary kinetic isotope effect for flavin reduction in MR and PETN reductase by nicotinamide coenzyme indicates that quantum mechanical tunneling plays a major role in hydride transfer. In PETN reductase, the kinetic isotope effect (KIE) is essentially independent of temperature in the experimentally accessible range, contrasting with strongly temperaturedependent reaction rates, consistent with a tunneling mechanism from the vibrational ground state of the reactive C-H/D bond. In MR, both the reaction rates and the KIE are dependent on temperature, and analysis using the Eyring equation suggests that hydride transfer has a major tunneling component, which, unlike PETN reductase, is gated by thermally induced vibrations in the protein. The oxidative half-reaction of MR is fully rate-limiting in steady-state turnover with the substrate 2-cyclohexenone and NADH at saturating concentrations. The KIE for hydride transfer from reduced flavin to the ␣/␤ unsaturated bond of 2-cyclohexenone is independent of temperature, contrasting with strongly temperaturedependent reaction rates, again consistent with groundstate tunneling. A large solvent isotope effect (SIE) accompanies the oxidative half-reaction, which is also independent of temperature in the experimentally accessible range. Double isotope effects indicate that hydride transfer from the flavin N5 atom to 2-cyclohexenone, and the protonation of 2-cyclohexenone, are concerted and both the temperature-independent KIE and SIE suggest that this reaction also proceeds by ground-state quantum tunneling. Our results demonstrate the importance of quantum tunneling in the reduction of flavins by nicotinamide coenzymes. This is the first observation of (i) three H-nuclei in an enzymic reaction being transferred by tunneling and (ii) the utilization of both passive and active dynamics within the same native enzyme.Enzymes are phenomenal catalysts that can achieve rate enhancements of up to ϳ21 orders of magnitude over the uncatalyzed reaction rate (1). Our quest to understand the physical basis of this catalytic power is challenging and has involved sustained and intensive research efforts by many workers in the physical and life sciences (for recent reviews see Refs. 2-5). Recent years have witnessed new activity in this area and extended our theoretical understanding beyond the shortcomings of transition state theory (6) to include roles for protein "motion" (7, 8), low barrier hydrogen bonds (e.g. Refs. 9 -11), active site preorganization (e.g. reviewed in Refs. 4 and 12), and quantum mechanical tunneling (for recent reviews see Refs. 13-15). New theoretical frameworks incorporating quantum mechanical tunneling and protein motion are emerging to address the catalytic potency of enzymes. These invoke motion in the protein and/or subs...

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The crystal structure of the human electron transferring flavoprotein (ETF)⅐medium chain acyl-CoA dehydrogenase (MCAD) complex reveals a dual mode of protein-protein interaction, imparting both specificity and promiscuity in the interaction of ETF with a range of structurally distinct primary dehydrogenases. ETF partitions the functions of partner binding and electron transfer between (i) the recognition loop, which acts as a static anchor at the ETF⅐MCAD interface, and (ii) the highly mobile redox active FAD domain. Together, these enable the FAD domain of ETF to sample a range of conformations, some compatible with fast interprotein electron transfer. Disorders in amino acid or fatty acid catabolism can be attributed to mutations at the protein-protein interface. Crucially, complex formation triggers mobility of the FAD domain, an induced disorder that contrasts with general models of protein-protein interaction by induced fit mechanisms. The subsequent interfacial motion in the MCAD⅐ETF complex is the basis for the interaction of ETF with structurally diverse protein partners. Solution studies using ETF and MCAD with mutations at the protein-protein interface support this dynamic model and indicate ionic interactions between MCAD Glu 212 and ETF Arg␣ 249 are likely to transiently stabilize productive conformations of the FAD domain leading to enhanced electron transfer rates between both partners.

The initial step in the l-kynurenine pathway is oxidation of l-tryptophan to N-formylkynurenine and is catalyzed by one of two heme enzymes, tryptophan 2,3-dioxygenase (TDO) or indoleamine 2,3-dioxygenase (IDO). Here, we address the role of the conserved active site Ser167 residue in human IDO (S167A and S167H variants), which is replaced with a histidine in other mammalian and bacterial TDO enzymes. Our kinetic and spectroscopic data for S167A indicate that this residue is not essential for O 2 or substrate binding, and we propose that hydrogen bond stabilization of the catalytic ferrous-oxy complex involves active site water molecules in IDO. The data for S167H show that the ferrous-oxy complex is dramatically destabilized in this variant, which is similar to the behavior observed in human TDO [Basran et al. (2008) Biochemistry 47, 4752-4760], and that this destabilization essentially destroys catalytic activity. New kinetic data for the wild-type enzyme also identify the ternary [enzyme-O 2-substrate] complex. The data reveal significant differences between the IDO and TDO enzymes, and the implications of these results are discussed in terms of our current understanding of IDO and TDO catalysis.

C-H bond breakage by tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH)occurs by vibrationally assisted tunneling (Basran, J., Sutcliffe, M. J., and Scrutton, N. S. (1999) Biochemistry 38, 3218 -3222). We show here a similar mechanism in TTQ-dependent aromatic amine dehydrogenase (AADH). The rate of TTQ reduction by dopamine in AADH has a large, temperature independent kinetic isotope effect (KIE ‫؍‬ 12.9 ؎ 0.2), which is highly suggestive of vibrationally assisted tunneling. H-transfer is compromised with benzylamine as substrate and the KIE is deflated (4.8 ؎ 0.2). The KIE is temperature-independent, but reaction rates are strongly dependent on temperature. With tryptamine as substrate reaction rates can be determined only at low temperature as C-H bond cleavage is rapid, and an exceptionally large KIE (54.7 ؎ 1.0) is observed. Studies with deuterated tryptamine suggest vibrationally assisted tunneling is the mechanism of deuterium and, by inference, hydrogen transfer. Bond cleavage by MADH using a slow substrate (ethanolamine) occurs with an inflated KIE (14.7 ؎ 0.2 at 25°C). The KIE is temperature-dependent, consistent with differential tunneling of protium and deuterium. Our observations illustrate the different modes of Htransfer in MADH and AADH with fast and slow substrates and highlight the importance of barrier shape in determining reaction rate. Aromatic amine dehydrogenase (AADH)1 and methylamine dehydrogenase (MADH) catalyze the oxidative deamination of primary amines to their corresponding aldehydes and ammonia.The physiological electron acceptors of MADH are amicyanin (for the Paracoccus denitrificans enzyme; Refs. 1-3) or a c-type cytochrome (for the Methylophilus methylotrophus enzyme; Ref. 4). AADH transfers electrons (derived from the deamination of aromatic amines) to the copper protein azurin (5). In a reductive half-reaction, both enzymes reduce the prosthetic group tryptophan tryptophylquinone (TTQ) through the initial formation of a carbinolamine intermediate ( Fig. 1; Refs. 5 and 6); loss of water from this carbinolamine produces an iminoquinone. The iminoquinone decays by breakage of the substrate C-H bond. An active site base that abstracts a proton from the substrate initiates bond cleavage (7). This step in the reductive half-reaction is rate-limiting, as demonstrated by the unusually large kinetic isotope effect (KIE) observed with methylamine (MADH; Refs. 8 -10) and dopamine (AADH; Ref. 11).Previously we demonstrated that cleavage of the substrate C-H bond of M. methylotrophus (sp. W 3 A 1 ) MADH is associated with a large KIE (16.8 Ϯ 0.5) and that the KIE is independent of temperature (8). Through detailed temperature dependence studies, we were able to suggest that C-H bond breakage occurs by extreme quantum tunneling (8). Quantum tunneling effects in enzymatic hydrogen transfer have been observed in only a small number of enzyme systems (8, 12-21). Most have been modeled using the classical formulations of transition state theory, incorporating a tunne...