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.     

Site-directed mutagenesis of proline 52 to glycine in amicyanin converts a true electron transfer reaction into one that is conformationally gated

Amicyanin is a type I copper protein that is the natural electron acceptor for the quinoprotein methylamine dehydrogenase (MADH). The conversion of Proline52 of amicyanin to a glycine does not alter the physical and spectroscopic properties of the copper binding site, but it does alter the rate of electron transfer (ET) from MADH. The values of electronic coupling (H(AB)) and reorganization energy (lambda) that are associated with the true ET reaction from the reduced O-quinol tryptophan tryptophylquinone (TTQ) of MADH to oxidized amicyanin are significantly altered as a consequence of the P52G mutation. The experimentally determined H(AB) increases from 12 to 78 cm(-1), and lambda increases from 2.3 to 2.8 eV. The rate and salt-dependence of the proton transfer-gated ET reaction from N-quinol MADH to amicyanin are also changed by the P52G mutation. Kinetic data suggests that a new common reaction step has become rate-limiting for both the true and gated ET reactions that occur from different redox forms of MADH. A comparison of the crystal structures of P52G amicyanin with those of native amicyanin free and in complex with MADH provided clues as to the basis for the change in ET parameters. The mutation results in the loss of three carbons from Pro52 and the movement of the neighboring residue Met51. This reduces the number of hydrophobic interactions with MADH in the complex and perturbs the protein-protein interface. A model is proposed for the ET reaction with P52G amicyanin in which the most stable conformation of the protein-protein complex with MADH is not optimal for ET. A new preceding kinetic step is introduced prior to true ET that requires P52G amicyanin to switch from this redox-inactive stable complex to a redox-active unstable complex. Thus, the ET reaction of P52G amicyanin is no longer a true ET but one that is conformationally gated by the reorientation of the proteins within the ET protein complex. This same reaction step now also gates the ET from N-quinol MADH, which is normally rate-limited by a proton transfer.     

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.     

A single methionine residue dictates the kinetic mechanism of interprotein electron transfer from methylamine dehydrogenase to amicyanin

Amicyanin is a type 1 copper protein that is the natural electron acceptor for the quinoprotein methylamine dehydrogenase (MADH). A P52G amicyanin mutation increased the Kd for complex formation and caused the normally true electron transfer (ET) reaction from O-quinol MADH to amicyanin to become a gated ET reaction (Ma, J. K., Carrell, C. J., Mathews, F. S., and Davidson, V. L. (2006) Biochemistry 45, 8284-8293). One consequence of the P52G mutation was to reposition the side chain of Met51, which is present at the MADH-amicyanin interface. To examine the precise role of Met51 in this interprotein ET reaction, Met51 was converted to Ala, Lys, and Leu. The Kd for complex formation of M51A amicyanin was unchanged but the experimentally determined electronic coupling increased from 12 cm-1 to 142 cm-1, and the reorganization energy increased from 2.3 to 3.1 eV. The rate and salt dependence of the proton transfer-gated ET reaction from N-quinol MADH to amicyanin is also changed by the M51A mutation. These changes in ET parameters and rates for the reactions with M51A amicyanin were similar to those caused by the P52G mutation and indicated that the ET reaction had become gated by a similar process, most likely a conformational rearrangement of the protein ET complex. The results of the M51K and M51L mutations also have consequences on the kinetic mechanism of regulation of the interprotein ET with effects that are intermediate between what is observed for the reaction of the native amicyanin and M51A amicyanin. These data indicate that the loss of the interactions involving Pro52 were primarily responsible for the change in Kd for P52G amicyanin, while the interactions involving the Met51 side chain are entirely responsible for the change in ET parameters and conversion of the true ET reaction of native amicyanin into a conformationally gated ET reaction.     

Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine dehydrogenase of Alcaligenes faecalis. Cofactor assembly and catalytic properties of recombinant enzyme expressed in Paracoccus denitrificans

The heterologous expression of tryptophan trytophylquinone (TTQ)-dependent aromatic amine dehydrogenase (AADH) has been achieved in Paracoccus denitrificans. The aauBEDA genes and orf-2 from the aromatic amine utilization (aau) gene cluster of Alcaligenes faecalis were placed under the regulatory control of the mauF promoter from P. denitrificans and introduced into P. denitrificans using a broad-host-range vector. The physical, spectroscopic and kinetic properties of the recombinant AADH were indistinguishable from those of the native enzyme isolated from A. faecalis. TTQ biogenesis in recombinant AADH is functional despite the lack of analogues in the cloned aau gene cluster for mauF, mauG, mauL, mauM and mauN that are found in the methylamine utilization (mau) gene cluster of a number of methylotrophic organisms. Steady-state reaction profiles for recombinant AADH as a function of substrate concentration differed between 'fast' (tryptamine) and 'slow' (benzylamine) substrates, owing to a lack of inhibition by benzylamine at high substrate concentrations. A deflated and temperature-dependent kinetic isotope effect indicated that C-H/C-D bond breakage is only partially rate-limiting in steady-state reactions with benzylamine. Stopped-flow studies of the reductive half-reaction of recombinant AADH with benzylamine demonstrated that the KIE is elevated over the value observed in steady-state turnover and is independent of temperature, consistent with (a) previously reported studies with native AADH and (b) breakage of the substrate C-H bond by quantum mechanical tunnelling. The limiting rate constant (k(lim)) for TTQ reduction is controlled by a single ionization with pK(a) value of 6.0, with maximum activity realized in the alkaline region. Two kinetically influential ionizations were identified in plots of k(lim)/K(d) of pK(a) values 7.1 and 9.3, again with the maximum value realized in the alkaline region. The potential origin of these kinetically influential ionizations is discussed.     

Catalysis by the isolated tryptophan tryptophylquinone-containing subunit of aromatic amine dehydrogenase is distinct from native enzyme and synthetic model compounds and allows further probing of TTQ mechanism

Para-substituted benzylamines are poor reactivity probes for structure-reactivity studies with TTQ-dependent aromatic amine dehydrogenase (AADH). In this study, we combine kinetic isotope effects (KIEs) with structure-reactivity studies to show that para-substituted benzylamines are good reactivity probes of TTQ mechanism with the isolated TTQ-containing subunit of AADH. Contrary to the TTQ-containing subunit of methylamine dehydrogenase (MADH), which is catalytically inactive, the small subunit of AADH catalyzes the oxidative deamination of a variety of amine substrates. Observed rate constants are second order with respect to substrate and inhibitor (phenylhydrazine) concentration. Kinetic studies with para-substituted benzylamines and their dideuterated counterparts reveal KIEs (>6) larger than those observed with native AADH (KIEs approximately unity). This is attributed to formation of the benzylamine-derived iminoquinone requiring structural rearrangement of the benzyl side chain in the active site of the native enzyme. This structural reorganization requires motions from the side chains of adjacent residues (which are absent in the isolated small subunit). The position of Phealpha97 in particular is responsible for the conformational gating (and hence deflated KIEs) observed with para-substituted benzylamines in the native enzyme. Hammett plots for the small subunit exhibit a strong correlation of structure-reactivity data with electronic substituent effects for para-substituted benzylamines and phenethylamines, unlike native AADH for which a poor correlation is observed. TTQ reduction in the isolated subunit is enhanced by electron withdrawing substituents, contrary to structure-reactivity studies reported for synthetic TTQ model compounds in which rate constants are enhanced by electron donating substituents. We infer that para-substituted benzylamines are good reactivity probes of TTQ mechanism with the isolated small subunit. This is attributed to the absence of structural rearrangement prior to H-transfer that limits the rate of TTQ reduction by para-substituted benzylamines in native enzyme.     

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.     

Site-directed mutagenesis of proline 94 to alanine in amicyanin converts a true electron transfer reaction into one that is kinetically coupled

Amicyanin is a type I copper protein that mediates electron transfer (ET) from methylamine dehydrogenase (MADH) to cytochrome c-551i. Pro(94) resides in the "ligand loop" of amicyanin, a sequence of amino acids that contains three of the four copper ligands. ET from the reduced O-quinol tryptophan tryptophylquinone of MADH to oxidized P94A amicyanin is a true ET reaction that exhibits values of electronic coupling (H(AB)) and reorganization energy (lambda) that are the same as for the reaction of native amicyanin. In contrast, the parameters for the ET reaction from reduced P94A amicyanin to oxidized cytochrome c-551i have been significantly altered as a consequence of the mutation. These values of H(AB) and lambda are 8.3 cm(-)(1) and 2.3 eV, respectively, compared to values of 0.3 cm(-)(1) and 1.2 eV for the reaction of native reduced amicyanin. The crystal structure of reduced P94A amicyanin exhibits two alternate conformations with the positions of the copper 1.4 A apart [Carrell, C. J., Sun, D., Jiang, S., Davidson, V. L., and Mathews, F. S. (2004) Biochemistry 43, 9372-9380]. In one of these, conformation B, a water molecule has replaced Met(98) as a copper ligand, and the ET distance to the heme of the cytochrome is increased by 1.4 A. Analysis of these structures suggests that the true k(ET) for ET from the copper in conformation B to heme would be much less than for ET from conformation A. A novel kinetic mechanism is proposed to explain these data in which the reduction of Cu(2+) by methylamine dehydrogenase is a true ET reaction while the oxidation of Cu(1+) by cytochrome c-551i is kinetically coupled ET. By comparison of the temperature dependence of the observed rate of the coupled ET reaction from reduced P94A amicyanin to cytochrome c-551i with the predicted rates and temperature dependence for the true ET reaction from conformation A, it was possible to determine the K(eq) and values of DeltaH degrees and DeltaS degrees that are associated with the non-ET reaction that modulates the observed ET rate.     

Effects of engineering uphill electron transfer into the methylamine dehydrogenase-amicyanin-cytochrome c-551i complex

Within the methylamine dehydrogenase-amicyanin-cytochrome c-551i complex, electrons are transferred from tryptophan tryptophylquinone (TTQ) to heme via the type I copper center of amicyanin. Mutation of Pro94 of amicyanin to Phe increases the redox potential of the copper center within the protein complex by approximately 195 mV. This introduces a large energy barrier for the second electron transfer (ET) step in this three-protein ET chain. As a consequence of this mutation, the ET rate from TTQ to copper exhibits about a 6-fold increase and the ET rate from copper to heme exhibits about a 100-fold decrease. These changes in ET rate are consistent with the predictions of Marcus theory. Temperature dependence studies of these reactions indicate that the reorganization energies for the ET to and from the copper center are unchanged by the P94F mutation, despite the large change in redox potential that it causes. Steady-state kinetic studies indicate that despite the large energy barrier for the ET from copper to heme, methylamine-dependent reduction of heme by the three-protein complex with P94F amicyanin goes to completion. The turnover number for this steady-state reaction, however, is decreased 50-fold relative to that of the native complex. As a consequence of the P94F mutation, the rate constant for the unfavorable uphill ET reaction from copper to heme has become the rate-limiting step in the overall reaction. The evolutionary implications of the effects of this mutation on the function of this naturally occurring simple ET chain are discussed.     

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.     

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.     

Proline 96 of the copper ligand loop of amicyanin regulates electron transfer from methylamine dehydrogenase by positioning other residues at the protein-protein interface

Amicyanin is a type 1 copper protein that serves as an electron acceptor for methylamine dehydrogenase (MADH). The site of interaction with MADH is a "hydrophobic patch" of amino acid residues including those that comprise a "ligand loop" that provides three of the four copper ligands. Three prolines are present in this region. Pro94 of the ligand loop was previously shown to strongly influence the redox potential of amicyanin but not affinity for MADH or mechanism of electron transfer (ET). In this study Pro96 of the ligand loop was mutated. P96A and P96G mutations did not affect the spectroscopic or redox properties of amicyanin but increased the K(d) for complex formation with MADH and altered the kinetic mechanism for the interprotein ET reaction. Values of reorganization energy (λ) and electronic coupling (H(AB)) for the ET reaction with MADH were both increased by the mutation, indicating that the true ET reaction observed with native amicyanin was now gated by or coupled to a reconfiguration of the proteins within the complex. The crystal structure of P96G amicyanin was very similar to that of native amicyanin, but notably, in addition to the change in Pro96, the side chains of residues Phe97 and Arg99 were oriented differently. These two residues were previously shown to make contacts with MADH that were important for stabilizing the amicyanin-MADH complex. The values of K(d), λ, and H(AB) for the reactions of the Pro96 mutants with MADH are remarkably similar to those obtained previously for P52G amicyanin. Mutation of this proline, also in the hydrophobic patch, caused reorientation of the side chain of Met51, another reside that interacted with MADH and caused a change in the kinetic mechanism of ET from MADH. These results show that proline residues near the copper site play key roles in positioning other amino acid residues at the amicyanin-MADH interface not only for specific binding to the redox protein partner but also to optimize the orientation of proteins for interprotein ET.     

Isotope effects reveal that para-substituted benzylamines are poor reactivity probes of the quinoprotein mechanism for aromatic amine dehydrogenase

Structure-activity correlations have been employed previously in the mechanistic interpretation of TTQ-dependent amine dehydrogenases using a series of para-substituted benzylamines. However, by combining the use of kinetic isotope effects (KIEs) and crystallographic analysis, in conjunction with structure-reactivity correlation studies, we show that para-substituted benzylamines are poor reactivity probes for TTQ-dependent aromatic amine dehydrogenase (AADH). Stopped-flow kinetic studies of the reductive half-reaction, with para-substituted benzylamines and their dideuterated counterparts, demonstrate that C-H or C-D bond breakage is not fully rate limiting (KIEs approximately unity). Contrary to previous reports, Hammett plots exhibit a poor correlation of structure-reactivity data with electronic substituent effects for para-substituted benzylamines and phenylethylamines. Crystallographic studies of enzyme-substrate complexes reveal that the observed structure-reactivity correlations are not attributed to distinct binding modes for para-substituted benzylamines in the active site, although two binding sites for p-nitrobenzylamine are identified. We identify structural rearrangements, prior to the H-transfer step, which are likely to limit the rate of TTQ reduction by benzylamines. This work emphasizes (i) the need for caution when applying structure-activity correlations to enzyme-catalyzed reactions and (ii) the added benefit of using both isotope effects and structural analysis, in conjunction with structure-reactivity relationships, to study chemical steps in enzyme reaction cycles.     

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.     

Re-engineering monovalent cation binding sites of methylamine dehydrogenase: effects on spectral properties and gated electron transfer

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone (TTQ)-dependent enzyme that catalyzes the oxidative deamination of primary amines. Monovalent cations are known to affect the spectral properties of MADH and to influence the rate of the gated electron transfer (ET) reaction from substrate-reduced MADH to amicyanin. Two putative monovalent cation binding sites in MADH have been identified by X-ray crystallography [Labesse, G., Ferrari, D., Chen, Z.-W., Rossi, G.-L., Kuusk, V., McIntire, W. S., and Mathews, F. S. (1998) J. Biol. Chem. 273, 25703-25712]. One requires cation-pi interactions involving residue alpha Phe55. An alpha F55A mutation differentially affects these two monovalent cation-dependent phenomena. The apparent K(d) associated with spectral perturbations increases 10-fold. The apparent K(d) associated with enhancement of the gated ET reaction becomes too small to measure, indicating that either it has decreased more than 1000-fold or the mutation has caused a conformational change that eliminates the requirement for the cation for the gated ET. These results show that of the two binding sites revealed in the structure, cation binding to the distal site, which is stabilized by the cation-pi interactions, is responsible for the spectral perturbations. Cation binding to the proximal site, which is stabilized by several oxygen ligands, is responsible for the enhancement of the rate of gated ET. Another site-directed mutant, alpha F55E MADH, exhibited cation binding properties that were the same as those of the native enzyme, indicating that interactions with the carboxylate of Glu can effectively replace the cation-pi interactions with Phe in stabilizing monovalent cation binding to the distal site.     

Catalysis and electron transfer in protein crystals: the binary and ternary complexes of methylamine dehydrogenase with electron acceptors

Polarized absorption microspectrophotometry has been used to detect catalysis and intermolecular electron transfer in single crystals of two multiprotein complexes: (1) the binary complex between Paracoccus denitrificans methylamine dehydrogenase, which contains tryptophan-tryptophylquinone (TTQ) as a cofactor, and its redox partner, the blue copper protein amicyanin; (2) the ternary complex between the same two proteins and cytochrome c-551i. Continuous wave electron paramagnetic resonance has been used to compare the state of copper in polycrystalline powders of the two systems. While catalysis and intermolecular electron transfer from reduced TTQ to copper are too fast to be accessible to our measurements, heme reduction occurs over a period of several minutes. The observed rate constant is about four orders of magnitude lower than in solution. The analysis of the temperature dependence of this apparent constant provides values for the parameters H(AB), related to electronic coupling between the two centers, and lambda, the reorganizational energy, that are compatible with electron transfer being the rate-determining step. From these parameters and the known distance between copper and heme, it is possible to calculate the parameter beta, which depends on the nature of the intervening medium, obtaining a value typical of electron transfer across a protein matrix. These findings suggest that the ternary complex in solution might achieve a higher efficiency than the rigid crystal structure thanks to an as yet unidentified role of protein dynamics.     

Correlation of rhombic distortion of the type 1 copper site of M98Q amicyanin with increased electron transfer reorganization energy

Mutation of the axial Met ligand of the type 1 copper site of amicyanin to Ala or Gln yielded M98A amicyanin, which exhibits typical axial type 1 ligation geometry but with a water molecule providing the axial ligand, and M98Q amicyanin, which exhibits significant rhombic distortion of the type 1 site (Carrell, C. J., Ma, J. K., Antholine, W. E., Hosler, J. P., Mathews, F. S., and Davidson, V. L. (2007) Biochemistry 46, 1900-1912). Despite the change of the axial ligand, the M98Q and M98A mutations had little effect on the redox potential of copper. The true electron transfer (ET) reactions from O-quinol methylamine dehydrogenase to oxidized native and mutant amicyanins revealed that the M98A mutation had little effect on kET, but the M98Q mutation reduced kET 45-fold. Thermodynamic analysis of the latter showed that the decrease in kET was due to an increase of 0.4 eV in the reorganization energy (lambda) associated with the ET reaction to M98Q amicyanin. No change in the experimentally determined electronic coupling or ET distance was observed, confirming that the mutation had not altered the rate-determining step for ET and that this was still a true ET reaction. The basis for the increased lambda is not the nature of the atom that provides the axial ligand because each uses an oxygen from Gln in M98Q amicyanin and from water in M98A amicyanin. Comparisons of the distance of the axial copper ligand from the equatorial plane that is formed by the other three copper ligands in isomorphous crystals of native and mutant amicyanins at atomic resolution indicate an increase in distance from 0.20 A in the native to 0.42 A in M98Q amicyanin and a slight decrease in distance for M98A amicyanin. This correlates with the rhombic distortion caused by the M98Q mutation that is clearly evident in the EPR and visible absorption spectra of the protein and suggests that the extent of rhombicity of the type 1 copper site influences the magnitude of lambda.     

Characterization of Electron Tunneling and Hole Hopping Reactions between Different Forms of MauG and Methylamine Dehydrogenase within a Natural Protein Complex

Respiration, photosynthesis, and metabolism require the transfer of electrons through and between proteins over relatively long distances. It is critical that this electron transfer (ET) occur with specificity to avoid cellular damage, and at a rate that is sufficient to support the biological activity. A multistep hole hopping mechanism could, in principle, enhance the efficiency of long-range ET through proteins as it does in organic semiconductors. To explore this possibility, two different ET reactions that occur over the same distance within the protein complex of the diheme enzyme MauG and different forms of methylamine dehydrogenase (MADH) were subjected to kinetic and thermodynamic analysis. An ET mechanism of single-step direct electron tunneling from diferrous MauG to the quinone form of MADH is consistent with the data. In contrast, the biosynthetic ET from preMADH, which contains incompletely synthesized tryptophan tryptophylquinone, to the bis-Fe(IV) form of MauG is best described by a two-step hole hopping mechanism. Experimentally determined ET distances matched the distances determined from the crystal structure that would be expected for single-step tunneling and multistep hopping. Experimentally determined relative values of electronic coupling (H(AB)) for the two reactions correlated well with the relative H(AB) values predicted from computational analysis of the structure. The rate of the hopping-mediated ET reaction is also 10-fold greater than that of the single-step tunneling reaction despite a smaller overall driving force for the hopping-mediated ET reaction. These data provide insight into how the intervening protein matrix and redox potentials of the electron donor and acceptor determine whether the ET reaction proceeds via single-step tunneling or multistep hopping.     

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].