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The paralogues TRPV5 and TRPV6 belong to the vanilloid subfamily of the transient receptor potential (TRP) superfamily of ion channels, and both play an important role in overall Ca2+homeostasis. The functioning of the channels centers on a tightly controlled Ca2+-dependent feedback mechanism in which the direct binding of the universal Ca2+-binding protein calmodulin (CaM) to the channel’s C-terminal tail is required for channel inactivation. We have investigated this interaction at the atomic level and propose that under basal cellular Ca2+concentrations CaM is constitutively bound to the channel’s C-tail via CaM C-lobe only contacts. When the cytosolic Ca2+concentration increases charging the apo CaM N-lobe with Ca2+, the CaM:TRPV6 complex rearranges and the TRPV6 C-tail further engages the CaM N-lobe via a crucial interaction involving L707. In a cellular context, mutation of L707 significantly increased the rate of channel inactivation. Finally, we present a model for TRPV6 CaM-dependent inactivation, which involves a novel so-called “two-tail” mechanism whereby CaM bridges two TRPV6 monomers resulting in closure of the channel pore.

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are heme-containing enzymes that catalyze the O2-dependent oxidation of l-tryptophan (l-Trp) in biological systems. Although many decades have passed since their discovery, the mechanism of tryptophan oxidation has not been established. It has been widely assumed that IDO and TDO react using the same mechanism, although there is no evidence that they do. For IDO, a Compound II (ferryl) species accumulates in the steady state and is implicated in the mechanism; in TDO, no such species has ever been observed. In this paper, we examine the kinetics of tryptophan oxidation in TDO. We find no evidence for the accumulation of Compound II during TDO catalysis. Instead, a ternary [Fe(II)–O2, l-Trp] complex is detected under steady state conditions. The absence of a Compound II species in the steady state in TDO is not due to an intrinsic inability of the TDO enzyme to form ferryl heme, because Compound II can be formed directly through a different route in which ferrous heme is reacted with peroxide. We interpret the data to mean that the rate-limiting step in the IDO and TDO mechanisms is not the same.

It is now widely accepted that enzyme-catalysed C–H bond breakage occurs by quantum mechanical tunnelling. This paradigm shift in the conceptual framework for these reactions away from semi-classical transition state theory (TST, i.e. including zero-point energy, but with no tunnelling correction) has been driven over the recent years by experimental studies of the temperature dependence of kinetic isotope effects (KIEs) for these reactions in a range of enzymes, including the tryptophan tryptophylquinone-dependent enzymes such as methylamine dehydrogenase and aromatic amine dehydrogenase, and the flavoenzymes such as morphinone reductase and pentaerythritol tetranitrate reductase, which produced observations that are also inconsistent with the simple Bell-correction model of tunnelling. However, these data—especially, the strong temperature dependence of reaction rates and the variable temperature dependence of KIEs—are consistent with other tunnelling models (termed full tunnelling models), in which protein and/or substrate fluctuations generate a configuration compatible with tunnelling. These models accommodate substrate/protein (environment) fluctuations required to attain a configuration with degenerate nuclear quantum states and, when necessary, motion required to increase the probability of tunnelling in these states. Furthermore, tunnelling mechanisms in enzymes are supported by atomistic computational studies performed within the framework of modern TST, which incorporates quantum nuclear effects.

TMADH (trimethylamine dehydrogenase) is a complex iron-sulphur flavoprotein that forms a soluble electron-transfer complex with ETF (electron-transferring flavoprotein). The mechanism of electron transfer between TMADH and ETF has been studied using stopped-flow kinetic and mutagenesis methods, and more recently by X-ray crystallography. Potentiometric methods have also been used to identify key residues involved in the stabilization of the flavin radical semiquinone species in ETF. These studies have demonstrated a key role for 'conformational sampling' in the electron-transfer complex, facilitated by two-site contact of ETF with TMADH. Exploration of three-dimensional space in the complex allows the FAD of ETF to find conformations compatible with enhanced electronic coupling with the 4Fe-4S centre of TMADH. This mechanism of electron transfer provides for a more robust and accessible design principle for interprotein electron transfer compared with simpler models that invoke the collision of redox partners followed by electron transfer. The structure of the TMADH-ETF complex confirms the role of key residues in electron transfer and molecular assembly, originally suggested from detailed kinetic studies in wild-type and mutant complexes, and from molecular modelling.

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.

An amperometric enzyme electrode was studied based on the wild-type protein trimethylamine dehydrogenase (TMADH), which catalyses the oxidative N-demethylation of trimethylamine to produce dimethylamine and formaldehyde. Ferrocene derivatives were investigated electrochemically, as free diffusing electron acceptors for recycling of the prosthetic groups of the immobilised enzyme. Ferricinium had the highest rates but, inhibited the enzyme, possibly as a result of a conformational change initiated at the Val-344 residue where it binds close to the 4Fe–4S cluster, interrupting the electron transfer between flavin mononucleotide (FMN) and 4Fe–4S by changing the redox potential of one or both of the prosthetic groups. (Dimethylamino)methylene ferrocene (DMAMFe) (k_s = 0.93 × 105 M−1 s−1) did not show inhibition and was used as a comparison for steady-state characterisation. The sensor response was studied over the pH range 6.0–11.0. Plots of k_cat/K_M revealed two ionisations with pK_a values of 7.5 and 10. The pK_a of 10 was attributed to the ionisation of the secondary amine in DMAMFe, whereas the pK_a of 7.5 was thought to reflect the ionisations of the intramolecular electron pathway. A TMADH/DMAMFe amperometric enzyme electrode was successfully used for the determination of TMA in different fish samples (detection limit: 2 mg TMA–N per 100g wet fish muscle). The obtained results compared well with a reference method based on picric acid.

A rational design for the site-specific immobilization of the protein trimethylamine dehydrogenase (TMADH) to facilitate charge transfer between enzyme and an electrode is described. Protein engineering and site-specific chemical modification have been used to extend the electron pathway from the protein surface to redox mediators. The kinetics of TMADH mutants (V344C and Y442C) modified with the iodoacetamide and 4-iodoacetamido 1-naphthole (IAN) showed that modification at position 344 has a more profound influence on intra- and inter-molecular electron transport, and the catalytic parameters k_cat and K_Mapp became a function of chemical modification. Ferricenium ion was shown to act as an electron acceptor for both mutants, but as its site of interaction is the residue 344, it was rejected for wiring in favour of [Fe(5-NH₂-phen)₃]2+, the latter showing similar very fast homogeneous electron exchange kinetics, ideal for ‘wire’ construction. The Y442C mutant was successfully immobilised on to an electrode surface which had been chemically modified with the redox polymer, poly-[Fe(5-NH₂-phen)₃]2+. This design enabled direct electrical communication between the enzyme and electrode. Using a partly oxidized polymer to limit the supply of oxidised electron acceptor, gave evidence for transition from the fast “0/2- cycle” to the “1/3-cycle” for the TMADH.

A rational design for the site-specific immobilization of the protein trimethylamine dehydrogenase TMADH to facilitate charge transfer between enzyme and an electrode is described. Protein engineering and site-specific chemical modification have been used to extend the electron pathway from the protein surface to redox mediators. The kinetics of TMADH mutants V344C and Y442C modified with the iodoacetamide and 4-iodoacetamido 1-naphthole IAN showed that modification at position 344 has a more profound influence on intra- and inter-molecular electron transport, and the catalytic parameters kcatand KMappbecame a function of chemical modification. Ferricenium ion was shown to act as an electron acceptor for both mutants, but as its site of interaction is the residue 344, it was rejected for wiring in favour of Fe5-NH2-phen32, the latter showing similar very fast homogeneous electron exchange kinetics, ideal for ‘wire’ construction. The Y442C mutant was successfully immobilised on to an electrode surface which had been chemically modified with the redox polymer, poly-Fe5-NH2-phen32. This design enabled direct electrical communication between the enzyme and electrode. Using a partly oxidized polymer to limit the supply of oxidised electron acceptor, gave evidence for transition from the fast “02- cycle” to the “13-cycle” for the TMADH.

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)Biochemistry38, 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 H-transfer in MADH and AADH with fast and slow substrates and highlight the importance of barrier shape in determining reaction rate.

The midpoint reduction potentials of the FAD cofactor in wild-type Methylophilus methylotrophus(sp. W3A1) electron-transferring flavoprotein (ETF) and the αR237A mutant were determined by anaerobic redox titration. The FAD reduction potential of the oxidized-semiquinone couple in wild-type ETF (E′1) is +153 ± 2 mV, indicating exceptional stabilization of the flavin anionic semiquinone species. Conversion to the dihydroquinone is incomplete (E′2< −250 mV), because of the presence of both kinetic and thermodynamic blocks on full reduction of the FAD. A structural model of ETF (Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett.5, 231–236) suggests that the guanidinium group of Arg-237, which is located over the siface of the flavin isoalloxazine ring, plays a key role in the exceptional stabilization of the anionic semiquinone in wild-type ETF. The major effect of exchanging αArg-237 for Ala in M. methylotrophusETF is to engineer a remarkable ∼200-mV destabilization of the flavin anionic semiquinone (E′2= −31 ± 2 mV, andE′1= −43 ± 2 mV). In addition, reduction to the FAD dihydroquinone in αR237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the αR237A ETF. Thus, kinetic (as well as thermodynamic) considerations are important in populating the redox forms of the protein-bound flavin. Additionally, we show that electron transfer from trimethylamine dehydrogenase to αR237A ETF is severely compromised, because of impaired assembly of the electron transfer complex.

Small angle x-ray solution scattering has been used to generate a low resolution, model-independent molecular envelope structure for electron-transferring flavoprotein (ETF) fromMethylophilus methylotrophus(sp. W3A1). Analysis of both the oxidized and 1-electron-reduced (anionic flavin semiquinone) forms of the protein revealed that the solution structures of the protein are similar in both oxidation states. Comparison of the molecular envelope of ETF from the x-ray scattering data with previously determined structural models of the protein suggests that ETF samples a range of conformations in solution. These conformations correspond to a rotation of domain II with respect to domains I and III about two flexible “hinge” sequences that are unique to M. methylotrophusETF. The x-ray scattering data are consistent with previous models concerning the interaction of M. methylotrophusETF with its physiological redox partner, trimethylamine dehydrogenase. Our data reveal that an “induced fit” mechanism accounts for the assembly of the trimethylamine dehydrogenase-ETF electron transfer complex, consistent with spectroscopic and modeling studies of the assembly process.

The reactions of wild-type trimethylamine dehydrogenase (TMADH) and of a His-172 Gln (H172Q) mutant were studied by rapid-mixing stopped-flow spectroscopy over the pH range 6.0-10.5, to address the potential role of His-172 in abstracting a proton from the substrate in a ‘carbanion’ mechanism for C-H bond cleavage. The pH-dependence of the limiting rate for flavin reduction (klim) was studied as a function of pH for the wild-type enzyme with perdeuterated trimethylamine as substrate. The use of perdeuterated trimethylamine facilitated the unequivocal identification of two kinetically influential ionizations in the enzyme-substrate complex, with macroscopic pKa values of 6.5±0.2 and 8.4±0.1. A plot of klim/Kd revealed a bell-shaped curve and two kinetically influential ionizations with macroscopic pKa values of 9.4±0.1 and 10.5±0.1. Mutagenesis of His-172, a potential active-site base and a component of a novel Tyr-His-Asp triad in the active site of TMADH, revealed that the pKa of 8.4±0.1 for the wild-type enzyme-substrate complex represents ionization of the imidazolium side-chain of His-172. H172Q TMADH retains catalytic competence throughout the pH range investigated. At pH 10.5, and in contrast with the wild-type enzyme, flavin reduction in H172Q TMADH is biphasic. The fast phase is dependent on the trimethylamine concentration and exhibits a kinetic isotope effect of about 3; C-H bond cleavage is thus partially rate-limiting. In contrast, the slow phase does not show hyperbolic dependence on substrate concentration, and the observed rate shows no dependence on isotope, revealing that C-H bond cleavage is not rate-limiting. The analysis of H172Q TMADH, together with data recently acquired for the Y169F mutant of TMADH, reveals that C-H bond breakage is not initiated via abstraction of a proton from the substrate by an active-site base. The transfer of reducing equivalents to flavin via a carbanion mechanism is therefore unlikely.

The reductive half-reaction of trimethylamine dehydrogenase with its physiological substrate trimethylamine has been examined by stopped-flow spectroscopy over the pH range 6.0–11.0, with attention focusing on the fastest of the three kinetic phases of the reaction, the flavin reduction/substrate oxidation process. As in previous work with the slow substrate diethylmethylamine, the reaction is found to consist of three well resolved kinetic phases. The observed rate constant for the fast phase exhibits hyperbolic dependence on the substrate concentration with an extrapolated limiting rate constant (klim) greater than 1000 s−1at pH above 8.5, 10 °C. The kinetic parameterklim/Kdfor the fast phase exhibits a bell-shaped pH dependence, with two pKavalues of 9.3 ± 0.1 and 10.0 ± 0.1 attributed to a basic residue in the enzyme active site and the ionization of the free substrate, respectively. The sigmoidal pH profile forklimgives a single pKavalue of 7.1 ± 0.2. The observed rate constants for both the intermediate and slow phases are found to decrease as the substrate concentration is increased. The steady-state kinetic behavior of trimethylamine dehydrogenase with trimethylamine has also been examined, and is found to be adequately described without invoking a second, inhibitory substrate-binding site. The present results demonstrate that: (a) substrate must be protonated in order to bind to the enzyme; (b) an ionization group on the enzyme is involved in substrate binding; (c) an active site general base is involved, but not strictly required, in the oxidation of substrate; (d) the fast phase of the reaction with native enzyme is considerably faster than observed with enzyme isolated from Methylophilus methylotrophusthat has been grown up on dimethylamine; and (e) a discrete inhibitory substrate-binding site is not required to account for excess substrate inhibition, the kinetic behavior of trimethylamine dehydrogenase can be readily explained in the context of the known properties of the enzyme.

Tyr-169 in trimethylamine dehydrogenase is one component of a triad also comprising residues His-172 and Asp-267. Its role in catalysis and in mediating the magnetic interaction between FMN cofactor and the 4Fe/4S center have been investigated by stopped-flow and EPR spectroscopy of a Tyr-169 to Phe (Y169F) mutant of the enzyme. Tyr-169 is shown to play an important role in catalysis (mutation to phenylalanine reduces the limiting rate constant for bleaching of the active site flavin by about 100-fold) but does not serve as a general base in the course of catalysis. In addition, we are able to resolve two kinetically influential ionizations involved in both the reaction of free enzyme with free substrate (as reflected inklim/Kd), and in the breakdown of the Eox·S complex (as reflected in klim). In EPR studies of the Y169F mutant, it is found that the ability of the Y169F enzyme to form the spin-interacting state between flavin semiquinone and reduced 4Fe/4S center characteristic of wild-type enzyme is significantly compromised. The present results are consistent with Tyr-169 representing the ionizable group of pKa∼9.5, previously identified in pH-jump studies of electron transfer, whose deprotonation must occur for the spin-interacting state to be established.

The ternary complex of Lactobacillus caseidihydrofolate reductase (DHFR) with folate and NADP+exists as a mixture of three interconverting forms (I, IIa and IIb) whose relative populations are pH dependent, with an effective pKof approx. 6. To investigate the role of Asp26in this pH dependence we have measured the 13C chemical shifts of [2,4a,7,9‐13C4]folate in its complex with the mutant DHFR Asp26→Asn and NADP+. Only a single form of the complex is detected and this has the characteristics of form I, an enol form with its N1 unprotonated. A study of the pH dependence of the 13C chemical shifts of DHFR selectively labelled with [4‐13C]aspartic acid in its complex with folate and NADP+indicates that no Asp residue has a pKvalue greater than 5.4. Two of the Asp CO−2signals appear as non‐integral signals with chemical shifts typical of non‐ionised COOH groups and with a pH dependence characteristic of the slow exchange equilibria previously characterised for signals in forms I and IIb (or IIa). It is proposed that the protonation/deprotonation controlling the equilibria involves the O4 position of the folate and that Asp26influences this indirectly by binding in its CO−2form to the protonated N1 group of folate in forms I and IIa thus reducing the pKinvolving protonation at the O4 position to approx. 6. These findings indicate that, in forms I and IIa of the ternary complex, folate binds to DHFR in a very similar way to methotrexate.

The ternary complex of Lactobacillus caseidihydrofolate reductase (DHFR) with folate and NADP +exists as a mixture of three interconverting forms (I, IIa and IIb) whose relative populations are pH dependent, with an effective p Kof approx. 6. To investigate the role of Asp 26in this pH dependence we have measured the 13C chemical shifts of [2,4 a,7,9- 13C 4]folate in its complex with the mutant DHFR Asp 26→Asn and NADP +. Only a single form of the complex is detected and this has the characteristics of form I, an enol form with its N1 unprotonated. A study of the pH dependence of the 13C chemical shifts of DHFR selectively labelled with [4- 13C]aspartic acid in its complex with folate and NADP +indicates that no Asp residue has a p Kvalue greater than 5.4. Two of the Asp CO −2signals appear as non-integral signals with chemical shifts typical of non-ionised COOH groups and with a pH dependence characteristic of the slow exchange equilibria previously characterised for signals in forms I and IIb (or IIa). It is proposed that the protonation/deprotonation controlling the equilibria involves the O4 position of the folate and that Asp 26influences this indirectly by binding in its CO −2form to the protonated N1 group of folate in forms I and IIa thus reducing the p Kinvolving protonation at the O4 position to approx. 6. These findings indicate that, in forms I and IIa of the ternary complex, folate binds to DHFR in a very similar way to methotrexate.