Active site aspartate residues are critical for tryptophan tryptophylquinone biogenesis in methylamine dehydrogenase

The biosynthesis of methylamine dehydrogenase (MADH) requires formation of six intrasubunit disulfide bonds, incorporation of two oxygens into residue betaTrp57 and covalent cross-linking of betaTrp57 to betaTrp108 to form the protein-derived cofactor tryptophan tryptophylquinone (TTQ). Residues betaAsp76 and betaAsp32 are located in close proximity to the quinone oxygens of TTQ in the enzyme active site. These residues are structurally conserved in quinohemoprotein amine dehydrogenase, which possesses a cysteine tryptophylquinone cofactor. Relatively conservative betaD76N and betaD32N mutations resulted in very low levels of MADH expression. Analysis of the isolated proteins by mass spectrometry revealed that each mutation affected TTQ biogenesis. betaD76N MADH possessed the six disulfides but had no oxygen incorporated into betaTrp57 and was completely inactive. The betaD32N MADH preparation contained a major species with six disulfides but no oxygen incorporated into betaTrp57 and a minor species with both oxygens incorporated, which was active. The steady-state kinetic parameters for the betaD32N mutant were significantly altered by the mutation and exhibited a 1000-fold increase in the Km value for methylamine. These results have allowed us to more clearly define the sequence of events that lead to TTQ biogenesis and to define novel roles for aspartate residues in the biogenesis of a protein-derived cofactor.     

Further insights into quinone cofactor biogenesis: probing the role of mauG in methylamine dehydrogenase tryptophan tryptophylquinone formation

Paracoccus denitrificans methylamine dehydrogenase (MADH) is an enzyme containing a quinone cofactor tryptophan tryptophylquinone (TTQ) derived from two tryptophan residues (betaTrp(57) and betaTrp(108)) within the polypeptide chain. During cofactor formation, the two tryptophan residues become covalently linked, and two carbonyl oxygens are added to the indole ring of betaTrp(57). Expression of active MADH from P. denitrificans requires four other genes in addition to those that encode the polypeptides of the MADH alpha(2)beta(2) heterotetramer. One of these, mauG, has been shown to be involved in TTQ biogenesis. It contains two covalently attached c-type hemes but exhibits unusual properties compared to c-type cytochromes and diheme cytochrome c peroxidases, to which it has some sequence similarity. To test the role that MauG may play in TTQ maturation, the predicted proximal histidine to each heme (His(35) and His(205)) has each been mutated to valine, and wild-type MADH was expressed in the background of these two mauG mutants. The resultant MADH has been characterized by mass spectrometry and electrophoretic and kinetic analyses. The majority species is a TTQ biogenesis intermediate containing a monohydroxylated betaTrp(57), suggesting that this is the natural substrate for MauG. Previous work has shown that MADH mutated at the betaTrp(108) position (the non-oxygenated TTQ partner) is predominantly also this intermediate, and work on these mutants is extended and compared to the MADH expressed in the background of the histidine to valine mauG mutations. In this study, it is unequivocally demonstrated that MauG is required to initiate the formation of the TTQ cross-link, the conversion of a single hydroxyl located on betaTrp(57) to a carbonyl, and the incorporation of the second oxygen into the TTQ ring to complete TTQ biogenesis. The properties of MauG, which are atypical of c-type cytochromes, are discussed in the context of these final stages of TTQ biogenesis.     

Kinetic and physical evidence that the diheme enzyme MauG tightly binds to a biosynthetic precursor of methylamine dehydrogenase with incompletely formed tryptophan tryptophylquinone

Methylamine dehydrogenase (MADH) contains the protein-derived cofactor tryptophan tryptophylquinone (TTQ) which is generated by the posttranslational modification of two endogenous tryptophan residues. The modifications are incorporation of two oxygens into one tryptophan side chain and the covalent cross-linking of that side chain to a second tryptophan residue. This process requires at least one accessory gene, mauG. Inactivation of mauG in vivo results in production of an inactive 119 kDa tetrameric alpha 2beta 2 protein precursor of MADH with incompletely synthesized TTQ. This precursor can be converted to active MADH with mature TTQ in vitro by reaction with MauG, a 42 kDa diheme enzyme. Steady-state kinetic analysis of the MauG-dependent conversion of the precursor to mature MADH with completely synthesized TTQ yielded values of k cat of 0.20 +/- 0.01 s (-1) and K m of 6.6 +/- 0.6 microM for the biosynthetic precursor protein in an in vitro assay. In the absence of an electron donor to initiate the reaction it was possible to isolate the MauG-biosynthetic precursor (enzyme-substrate) complex in solution using high-resolution size-exclusion chromatography. This stable complex is noncovalent and could be separated into its component proteins by anion-exchange chromatography. In contrast to the enzyme-substrate complex, a mixture of MauG and its reaction product, mature MADH, did not elute as a complex during size-exclusion chromatography. The differential binding of MauG to its protein substrate and protein product of the reaction indicates that significant conformational changes in one or both of the proteins occur during catalysis which significantly affects the protein-protein interactions.     

Aromatic amine dehydrogenase, a second tryptophan tryptophylquinone enzyme.

Aromatic amine dehydrogenase (AADH) catalyzes the oxidative deamination of aromatic amines including tyramine and dopamine. AADH is structurally similar to methylamine dehydrogenase (MADH) and possesses the same tryptophan tryptophylquinone (TTQ) prosthetic group. AADH exhibits an alpha 2 beta 2 structure with subunit molecular weights of 39,000 and 18,000 and with a quinone covalently attached to each beta subunit. Neither subunit cross-reacted immunologically with antibodies to the corresponding subunits of MADH, and the N-terminal amino acid sequence of the beta subunit of AADH exhibited no homology with the highly conserved beta subunits of MADH. The absorption spectra for the oxidized, semiquinone, and reduced forms of AADH have been characterized, and extinction coefficients for the absorption maxima of each redox form have been determined. These spectra are very similar to those for MADH, indicating the likelihood of a TTQ cofactor. This was verified by the near identity of the vibrational frequencies and intensities in the resonance Raman spectra for the oxidized forms of AADH and MADH. A stable semiquinone of AADH could be observed during a reductive titration with dithionite, whereas titration with tyramine proceeded directly from the oxidized to the reduced form. AADH was very stable against denaturation by heat and exposure to guanidine. The individual subunits could be separated by gel filtration after incubation in guanidine hydrochloride, and partial reconstitution of activity was observed on recombination of the subunits. Steady-state kinetic analysis of AADH yielded a Vmax of 17 mumol/min/mg and a Km for tyramine of 5.4 microM. Substrate inhibition by tyramine was observed. AADH was irreversibly inhibited by hydrazine, phenylhydrazine, hydroxylamine, semicarbazide, and aminoguanidine. Isonicotinic acid hydrazide (isoniazid) and isonicotinic acid 2-isopropyl hydrazide (iproniazid) were reversible noncompetitive inhibitors of AADH and exhibited K(i) values of 8 and 186 microM, respectively. The similarities and differences between AADH and other amine oxidizing enzymes are also discussed.     

Mutation of alphaPhe55 of methylamine dehydrogenase alters the reorganization energy and electronic coupling for its electron transfer reaction with amicyanin

Methylamine dehydrogenase (MADH) possesses an alpha(2)beta(2) structure with each smaller beta subunit possessing a tryptophan tryptophylquinone (TTQ) prosthetic group. Phe55 of the alpha subunit is located where the substrate channel from the enzyme surface opens into the active site. Site-directed mutagenesis of alphaPhe55 has revealed roles for this residue in determining substrate specificity and binding monovalent cations at the active site. It is now shown that the alphaF55A mutation also increases the rate of the true electron transfer (ET) reaction from O-quinol MADH to amicyanin. The reorganization energy associated with the ET reaction is decreased from 2.3 to 1.8 eV. The electronic coupling associated with the ET reaction is decreased from 12 to 3 cm(-1). The crystal structure of alphaF55A MADH in complex with its electron acceptors, amicyanin and cytochrome c-551i, has been determined. Little difference in the overall structure is seen, relative to the native complex; however, there are significant changes in the solvent content of the active site and substrate channel. The crystal structure of alphaF55A MADH has also been determined with phenylhydrazine covalently bound to TTQ in the active site. Phenylhydrazine binding significantly perturbs the orientation of the TTQ rings relative to each other. The ET results are discussed in the context of the new and old crystal structures of the native and mutant enzymes.     

Identification of a new reaction intermediate in the oxidation of methylamine dehydrogenase by amicyanin

The two-electron oxidation of tryptophan tryptophylquinone (TTQ) in substrate-reduced methylamine dehydrogenase (MADH) by amicyanin is known to proceed via an N-semiquinone intermediate in which the substrate-derived amino group remains covalently attached to TTQ [Bishop, G. R., and Davidson, V. L. (1996) Biochemistry 35, 8948-8954]. A new method for the stoichiometric formation of the N-semiquinone in vitro has allowed the study of the oxidation of the N-semiquinone by amicyanin in greater detail than was previously possible. Conversion of N-semiquinone TTQ to the quinone requires two biochemical events, electron transfer to amicyanin and release of ammonia from TTQ. Using rapid-scanning stopped-flow spectroscopy, it is shown that this occurs by a sequential mechanism in which oxidation to an imine (N-quinone) precedes hydrolysis by water and ammonia release. Under certain reaction conditions, the N-quinone intermediate accumulates prior to the relatively slow hydrolysis step. Correlation of these transient kinetic data with steady-state kinetic data indicates that the slow hydrolysis of the N-quinone by water does not occur in the steady state. In the presence of excess substrate, the next methylamine molecule initiates a nucleophilic attack of the N-quinone TTQ, causing release of ammonia that is concomitant with the formation of the next enzyme-substrate cofactor adduct. In light of these results, the usually accepted steady-state reaction mechanism of MADH is revised and clarified to indicate that reactions of the quinone form of TTQ are side reactions of the normal catalytic pathway. The relevance of these conclusions to the reaction mechanisms of other enzymes with carbonyl cofactors, the reactions of which proceed via Schiff base intermediates, is also discussed.     

Active-site residues are critical for the folding and stability of methylamine dehydrogenase.

Site-directed mutagenesis was used to alter active-site residues of methylamine dehydrogenase (MADH) from Paracoccus denitrificans. Four residues of the beta subunit of MADH which are in close proximity to the tryptophan tryptophylquinone (TTQ) prosthetic group were modified. The crystal structure of MADH reveals that each of these residues participates in hydrogen bonding interactions with other active-site residues, TTQ or water. Relatively conservative mutations which removed the potentially reactive oxygens on the side chains of Thr122, Tyr119, Asp76 and Asp32 each resulted in greatly reduced or undetectable levels of MADH production. The reduction of MADH levels was determined by assays of activity and Western blots of crude extracts with antisera specific for the MADH beta subunit. No activity or cross-reactive protein was detected in extracts of cells expressing D76N, T122A and T122C MADH mutants. Very low levels of active MADH were produced by cells expressing D32N, Y119F, Y119E and Y119K MADH mutants. The Y119F and D32N mutants were purified from cell extracts and found to be significantly less stable than wild-type MADH. Only the T122S MADH mutant was produced at near wild-type levels. Possible roles for these amino acid residues in stabilizing unusual structural features of the MADH beta subunit, protein folding and TTQ biosynthesis are discussed.     

Probing mechanisms of catalysis and electron transfer by methylamine dehydrogenase by site-directed mutagenesis of alpha Phe55

Methylamine dehydrogenase (MADH) possesses an alpha(2)beta(2) subunit structure with each smaller beta subunit possessing a tryptophan tryptophylquinone (TTQ) prosthetic group. Phe(55) of the alpha subunit is located where the substrate channel from the enzyme surface opens into the active site. Site-directed mutagenesis studies have revealed several roles for this residue in catalysis and electron transfer (ET) by MADH. Site-directed mutagenesis of either alpha Phe(55) or beta Ile(107) (a residue in the beta subunit which interacts with alpha Phe(55)) converts MADH into enzymes with specificities for long-chain amines, amylamine or propylamine. Mutation of alpha Phe(55) also affects monovalent cation binding to the active site. alpha F55A MADH exhibits an increased K(d) for cation-dependent spectral changes and a decreased K(d) for cation-dependent stimulation of the rate of gated ET from N-quinol MADH to amicyanin. These results demonstrate that alpha Phe(55) is able to directly participate in a wide range of biochemical processes not typically observed for a phenylalanine residue.     

Electron transfer in quinoproteins

Soluble quinoprotein dehydrogenases oxidize a wide range of sugar, alcohol, amine, and aldehyde substrates. The physiological electron acceptors for these enzymes are not pyridine nucleotides but are other soluble redox proteins. This makes these enzymes and their electron acceptors excellent systems with which to study mechanisms of long-range interprotein electron transfer reactions. The tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH) transfers electrons to a blue copper protein, amicyanin. It has been possible to alter the rate of electron transfer by using different redox forms of MADH, varying reaction conditions, and performing site-directed mutagenesis on these proteins. From kinetic and thermodynamic analyses of the reaction rates, it was possible to determine whether a change in rate is due a change in Delta G(0), electronic coupling, reorganization energy or kinetic mechanism. Examples of each of these cases are discussed in the context of the known crystal structures of the electron transfer protein complexes. The pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase transfers electrons to a c-type cytochrome. Kinetic and thermodynamic analyses of this reaction indicated that this electron transfer reaction was conformationally coupled. Quinohemoproteins possess a quinone cofactor as well as one or more c-type hemes within the same protein. The structures of a PQQ-dependent quinohemoprotein alcohol dehydrogenase and a TTQ-dependent quinohemoprotein amine dehydrogenase are described with respect to their roles in intramolecular and intermolecular protein electron transfer reactions.     

Conversion of methylamine dehydrogenase to a long-chain amine dehydrogenase by mutagenesis of a single residue

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone (TTQ) dependent enzyme that catalyzes the oxidative deamination of primary amines. Amino acid residues of both the TTQ-bearing beta subunit and the noncatalytic alpha subunit line a substrate channel that leads from the protein surface to the enzyme active site. Phe55 of the alpha subunit is located at the opening of the active site. Conversion of alphaPhe55 to alanine dramatically alters the substrate preference of MADH. The K(m) for methylamine increases from 9 microM to 15 mM. The preferred substrates are now primary amines with chain lengths of at least seven carbons. The K(m) for 1, 10-diaminodecane is 11 microM, compared to 1.2 mM for wild-type MADH. Despite the large variation in K(m) values, k(cat) values are relatively unaffected by the mutation. Molecular modeling of substrates into the crystal structure of the enzyme active site and substrate channel provides an explanation for the dramatic changes in substrate specificity caused by this mutation of a single amino acid residue.     

Characterization of the tryptophan-derived quinone cofactor of methylamine dehydrogenase by resonance Raman spectroscopy

The resonance Raman (RR) spectrum of oxidized methylamine dehydrogenase (MADHOX) exhibits a set of C-H, C-C, C = C, and C = O vibrational modes between 900 and 1700 cm-1 that are characteristic of the quinone moiety of the tryptophan tryptophlyquinone (TTQ) cofactor. The close similarity of the RR spectra for MADHs from Paracoccus denitrificans (Pd), Thiobacillus versutus (Tv), and bacterium W3A1 proves that the same cofactor is present in all three proteins. The MADHs from Pd and Tv have a v(C = O) mode at approximately 1625 cm-1 that shifts approximately 20 cm-1 upon 18O substitution of one of the carbonyl oxygens and is assigned to the in-phase symmetric stretch of the two C = O groups. The semiquinone form of Pd MADH has its own characteristic RR spectrum with altered peak frequencies and intensities as well as a decrease in the total number of peaks. The hydroxide and ammonia adducts of MADHOX produce RR spectra similar to that of the semiquinone. The spectral changes in all three cases are interpreted as being due to reduced conjugation of the cofactor. The ammonia adduct is formulated as a carbinolamine, a likely intermediate in the enzymatic mechanism. In contrast, formation of the electron-transfer complex between amicyanin and MADHOX has no effect on the vibrational frequencies (and, hence, structure) of either the MADH quinone or the amicyanin blue copper site. The behavior of the TTQ cofactors of Pd and Tv MADHs are very similar to one another and somewhat different from W3A1 MADH, particularly with regard to adduct formation and ability to undergo isotope exchange with solvent. These differences are ascribed to the cofactor environments within the proteins rather than to the structure of the cofactor itself.     

Redox properties of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans

Paracoccus denitrificans produces two primary enzymes for the amine oxidation, tryptophan-tryptophylquinone (TTQ)-containing methylamine dehydrogenase (MADH) and quinohemoprotein amine dehydrogenase (QH-AmDH). QH-AmDH has a novel cofactor, cysteine tryptophylquinone (CTQ) and two hemes c. In this work, the redox potentials of three redox centers in QH-AmDH were determined by a mediator-assisted continuous-flow column electrolytic spectroelectrochemical technique. Kinetics of the electron transfer from QH-AmDH to three kinds of metalloproteins, amicyanin, cytochrome c(550), and horse heart cytochrome c were examined on the basis of the theory of mediated-bioelectrocatalysis. All these metalloproteins work as a good electron acceptor of QH-AmDH and donate the electron to the terminal oxidase of P. denitrificans, which was revealed by reconstitution of the respiratory chain. These properties are in marked contrast with those of MADH, which shows high specificity to amicyanin. These electron transfer kinetics are discussed in terms of thermodynamics and structural property.     

Mechanistic possibilities in MauG-dependent tryptophan tryptophylquinone biosynthesis

Tryptophan tryptophylquinone (TTQ), the prosthetic group of methylamine dehydrogenase, is formed by post-translational modifications of two tryptophan residues that result in the incorporation of two oxygens into one tryptophan side chain and the covalent cross-linking of that side chain to a second tryptophan residue. MauG is a novel 42 kDa di-heme protein, which is required for the biosynthesis of TTQ. An experimental system has been developed that allows the direct continuous monitoring of MauG-dependent TTQ biosynthesis in vitro. Four diverse electron donors, ascorbate, dithiothreitol, reduced glutathione, and NADH, were each able to provide reducing equivalents for MauG-dependent TTQ biosynthesis under aerobic conditions. The reaction with NADH was mediated by an NADH-dependent oxidoreductase. Under anaerobic conditions in the absence of an electron donor, H(2)O(2) could serve as a substrate for MauG-dependent TTQ biosynthesis. During the reaction with H(2)O(2), a discrete reaction intermediate was observed, which is likely the reduced quinol form of TTQ that then is oxidized to the quinone. These results suggest that not only the incorporation of oxygen into the monohydroxylated biosynthetic intermediate but also the subsequent oxidation of quinol MADH during TTQ biosynthesis is a MauG-dependent process. The implications of these results in elucidating the mechanism of MauG-dependent TTQ biosynthesis and identifying potential physiologic electron and oxygen donors for TTQ biosynthesis in vivo are discussed.     

Crystallographic investigations of the tryptophan-derived cofactor in the quinoprotein methylamine dehydrogenase

A model of tryptophan tryptophylquinone (TTQ), recently proposed by McIntire et al. (Science (1991) 252, 817-824) to be the prosthetic group of the quinoprotein methylamine dehydrogenase, has been compared with electron density maps of this dehydrogenase from Thiobacillus versutus and Paracoccus denitrificans. The comparison shows that the TTQ model can be neatly accommodated, providing strong supportive evidence that TTQ is indeed the cofactor for this group of quinoproteins.     

Crystal structure of quinohemoprotein amine dehydrogenase from Pseudomonas putida. Identification of a novel quinone cofactor encaged by multiple thioether cross-bridges.

The crystal structure of a quinohemoprotein amine dehydrogenase from Pseudomonas putida has been determined at 1.9-A resolution. The enzyme comprises three non-identical subunits: a four-domain alpha-subunit that harbors a di-heme cytochrome c, a seven-bladed beta-propeller beta-subunit that provides part of the active site, and a small gamma-subunit that contains a novel cross-linked, proteinous quinone cofactor, cysteine tryptophylquinone. More surprisingly, the catalytic gamma-subunit contains three additional chemical cross-links that encage the cysteine tryptophylquinone cofactor, involving a cysteine side chain bridged to either an Asp or Glu residue all in a hitherto unknown thioether bonding with a methylene carbon atom of acidic amino acid side chains. Thus, the structure of the 79-residue gamma-subunit is quite unusual, containing four internal cross-links in such a short polypeptide chain that would otherwise be difficult to fold into a globular structure.     

Tryptophan tryptophylquinone biosynthesis: a radical approach to posttranslational modification

Protein-derived cofactors are formed by irreversible covalent posttranslational modification of amino acid residues. An example is tryptophan tryptophylquinone (TTQ) found in the enzyme methylamine dehydrogenase (MADH). TTQ biosynthesis requires the cross-linking of the indole rings of two Trp residues and the insertion of two oxygen atoms onto adjacent carbons of one of the indole rings. The diheme enzyme MauG catalyzes the completion of TTQ within a precursor protein of MADH. The preMADH substrate contains a single hydroxyl group on one of the tryptophans and no crosslink. MauG catalyzes a six-electron oxidation that completes TTQ assembly and generates fully active MADH. These oxidation reactions proceed via a high valent bis-Fe(IV) state in which one heme is present as Fe(IV)=O and the other is Fe(IV) with both axial heme ligands provided by amino acid side chains. The crystal structure of MauG in complex with preMADH revealed that catalysis does not involve direct contact between the hemes of MauG and the protein substrate. Rather it is accomplished through long-range electron transfer, which presumably generates radical intermediates. Kinetic, spectrophotometric, and site-directed mutagenesis studies are beginning to elucidate how the MauG protein controls the reactivity of the hemes and mediates the long range electron/radical transfer required for catalysis. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

Crystal structure of an electron transfer complex between aromatic amine dehydrogenase and azurin from Alcaligenes faecalis

The crystal structure of an electron transfer complex of aromatic amine dehydrogenase (AADH) and azurin is presented. Electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of AADH to the type I copper of the cupredoxin azurin. This structure is compared with the complex of the TTQ-containing methylamine dehydrogenase (MADH) and the cupredoxin amicyanin. Despite significant similarities between the two quinoproteins and the two cupredoxins, each is specific for its respective partner and the ionic strength dependence and magnitude of the binding constant for each complex are quite different. The AADH-azurin interface is largely hydrophobic, covering approximately 500 A(2) of surface on each molecule, with one direct hydrogen bond linking them. The closest distance from TTQ to copper is 12.6 A compared with a distance of 9.3 A in the MADH-amicyanin complex. When the MADH-amicyanin complex is aligned with the AADH-azurin complex, the amicyanin lies on top of the azurin but is oriented quite differently. Although the copper atoms differ in position by approximately 4.7 A, the amicyanin bound to MADH appears to be rotated approximately 90 degrees from its aligned position with azurin. Comparison of the structures of the two complexes identifies features of the interface that dictate the specificity of the protein-protein interaction and determine the rate of interprotein electron transfer.     

Catching catalysis in the act: using single crystal kinetics to trap methylamine dehydrogenase reaction intermediates

Methylamine dehydrogenase (MADH) is produced by a range of gram-negative methylotrophic and autotrophic bacteria, and allows the organisms to utilise methylamine as the sole source of carbon. The enzyme catalyses the oxidation of methylamine to formaldehyde and ammonia, leaving it in a two-electron reduced state. To complete the catalytic cycle, MADH is reoxidised via an electron transfer (ET) chain. The redox center in the enzyme is the organic cofactor tryptophan tryptophylquinone (TTQ) derived from the posttranslational modification of two Trp residues in the protein. This cofactor has spectral features in the visible region, which change during catalytic turnover, defining spectrally distinct reaction intermediates that reflect the electronic state of the TTQ. In the case of the Paracoccus denitrificans enzyme the physiologic ET chain involves the protein redox partner amicyanin (a blue copper protein). A stable binary (MADH/amicyanin) complex can be formed, and its crystal structure has been solved to 2.5 A resolution by Chen et al. [Biochemistry 21 (1992) 4959]. These crystals were shown to be competent for catalysis and ET by Merli et al. [J. Biol. Chem. 271 (1996) 9177] using single crystal polarised absorption spectroscopy. Through a novel combination of single crystal visible microspectrophotometry, X-ray crystallography and freeze-trapping, we have trapped reaction intermediates of the enzyme in complex with its physiological redox partner amicyanin in the crystalline state. We will present data confirming that catalysis and ET in the binary complex crystals can be tracked by single crystal visible microspectrophotometry. We will also show that the reaction pathway is unperturbed by the presence of cryoprotectant solution, enabling direct freeze-trapping of reaction intermediates within the crystal. We will present new data demonstrating that the binary complex crystals are also capable of exhibiting UV light-dependent oxidase activity, as observed in solution [Biochim. Biophys. Acta 1364 (1998) 297].     

Atomic level insight into the oxidative half-reaction of aromatic amine dehydrogenase

The quinoprotein aromatic amine dehydrogenase (AADH) uses a covalently bound tryptophan tryptophylquinone (TTQ) cofactor to oxidatively deaminate primary aromatic amines. Recent crystal structures have provided insight into the reductive half-reaction. In contrast, no atomic details are available for the oxidative half-reaction. The TTQ O7 hydroxyl group is protonated during reduction, but it is unclear how this proton can be removed during the oxidative half-reaction. Furthermore, compared with the electron transfer from the N-quinol form, electron transfer from the non-physiological O-quinol form to azurin is significantly slower. Here we report crystal structures of the O-quinol, N-quinol, and N-semiquinone forms of AADH. A comparison of oxidized and substrate reduced AADH species reveals changes in the TTQ-containing subunit, extending from residues in the immediate vicinity of the N-quinol to the putative azurin docking site, suggesting a mechanism whereby TTQ redox state influences interprotein electron transfer. In contrast, chemical reduction of the TTQ center has no significant effect on protein conformation. Furthermore, structural reorganization upon substrate reduction places a water molecule near TTQ O7 where it can act as proton acceptor. The structure of the N-semiquinone, however, is essentially similar to oxidized AADH. Surprisingly, in the presence of substrate a covalent N-semiquinone substrate adduct is observed. To our knowledge this is the first detailed insight into a complex, branching mechanism of quinone oxidation where significant structural reorganization upon reduction of the quinone center directly influences formation of the electron transfer complex and nature of the electron transfer process.     

Importance of barrier shape in enzyme-catalyzed reactions. Vibrationally assisted hydrogen tunneling in tryptophan tryptophylquinone-dependent amine dehydrogenases

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 degrees 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.