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Highlights: [•] LodA has a tryptophylquinone cofactor but functions as an oxidase not a dehydrogenase. [•] LodA exhibits a ping-pong kinetic mechanism with lysine and O2 as substrates. [•] The kinetic mechanism of LodA is consistent with a covalent lysine-CTQ intermediate. [ABSTRACT FROM AUTHOR]

Abstract: Tryptophylquinone cofactors are formed by posttranslational modifications that result in the incorporation of two oxygens into a tryptophan side chain, and the covalent cross-linking of that side chain to another amino acid residue. Tryptophylquinone enzymes catalyze the oxidative deamination of primary amines, and utilize other redox proteins as electron acceptors. Mechanistic and structural studies of these enzymes are providing insight into how these enzymes utilize these highly reactive protein-derived quinones in a controlled manner to facilitate biologically important catalytic and electron transfer reactions. [Copyright &y& Elsevier]

Methylamine dehydrogenase (MADH) possesses an α2β2 subunit structure with each smaller β subunit possessing a tryptophan tryptophylquinone (TTQ) prosthetic group. Phe55 of the α 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 αPhe55 or βIle107 (a residue in the β subunit which interacts with αPhe55) converts MADH into enzymes with specificities for long-chain amines, amylamine or propylamine. Mutation of αPhe55 also affects monovalent cation binding to the active site. αF55A MADH exhibits an increased Kd for cation-dependent spectral changes and a decreased Kd for cation-dependent stimulation of the rate of gated ET from N-quinol MADH to amicyanin. These results demonstrate that αPhe55 is able to directly participate in a wide range of biochemical processes not typically observed for a phenylalanine residue. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Examines the re-engineering of monovalent cation binding sites of methylamine dehydrogenase (MADH). Characteristics of MADH; Effect of monovalent cations on the spectral properties of MADH; Rate of the gated electron transfer reaction from substrate-reduced MADH to amicyanin; Identification of two putative monovalent cation binding sites in MADH.

Identifies a novel reaction intermediate in the oxidation of methylamine dehydrogenase by amicyanin. Stoichiometric formation of the N-semiquinone in vitro; Electron transfer to amicyanin; Release of ammonia from tryptophan tryptophylquinone (TTQ); Initiation of nucleophilic attack of the N-quinone TTQ by the methylamine molecule.

Presents a study that examined the reaction of trimethylamine dehydrogenase (TMADH) with trimethylamine. How the reaction was examined; Basis of study; Findings.

Examines the oxidation of methanol dehydrogenase by cytochrome as a function of ionic strength with the use of stopped-flow spectroscopy. Dissociation constants; Rate constants for the electron transfer reaction; Oxidation model of methanol dehydrogenase.

Presents kinetic and thermodynamic results of binding and electron transfer reactions between methanol dehydrogenase and its physiologic electron acceptor cytochrome c-551i. Electronic coupling; Reorganizational energy; Utilization of Paracoccus denitrificans.

Presents results from experiments that study the nature of the rate-limiting proton transfer (PT) step which gates electron transfer (ET) from N-quinol tryptophan tryptophylquinone (TTQ) of methylamine dehydrogenase (MADH) to amicyanin. Analysis of transient kinetic data; Dependence of the rates of O- and N-quinol oxidation on monovalent cations.

Probes the roles of specific amino acid residues of amicyanin in stabilizing the methylamine dehydrogenase (MADH)-amicyanin complex and determines the ionic strength dependence of complex formation. Expression of saturation behavior through biphasic kinetics; Rationale for site-directed mutagenesis experiment; Specificity of MADH for amicyanin.

GoxA is a glycine oxidase that possesses a cysteine tryptophylquinone (CTQ) cofactor that is formed by posttranslational modifications that are catalyzed by a modifying enzyme GoxB. It is the second known tryptophylquinone enzyme to function as an oxidase, the other being the lysine -oxidase, LodA. All other enzymes containing CTQ or tryptophan tryptophylquinone (TTQ) cofactors are dehydrogenases. Kinetic analysis of GoxA revealed allosteric cooperativity for its glycine substrate, but not O2. This is the first CTQ- or TTQ-dependent enzyme to exhibit cooperativity. Here, we show that cooperativity and homodimer stabilization are strongly dependent on the presence of Phe-237. Conversion of this residue, which is a Tyr in LodA, to Tyr or Ala eliminates the cooperativity and destabilizes the dimer. These mutations also significantly affect the kcat and Km values for the substrates. On the basis of structural and modeling studies, a mechanism by which Phe-237 exerts this influence is presented. Two active site residues, Asp-547 and His-466, were also examined and shown by site-directed mutagenesis to be critical for CTQbiogenesis. This result is compared with the results of similar studies of mutagenesis of structurally conserved residues of other tryptophylquinone enzymes. These results provide insight into the roles of specific active-site residues in catalysis and CTQ biogenesis, as well as describing an interesting mechanism by which a single residue can dictate whether or not an enzyme exhibits cooperative allosteric behavior toward a substrate. [ABSTRACT FROM AUTHOR]

MauG is a diheme enzyme responsible for the post-translational formation of the catalytic tryptophan tryptophylquinone (TTQ) cofactor in methylamine dehydrogenase (MADH). MauG can utilize hydrogen peroxide, or molecular oxygen and reducing equivalents, to complete this reaction via a catalytic bis-Fe(IV) intermediate. Crystal structures of diferrous, Fe(II)-CO, and Fe(II)-NO forms of MauG in complex with its preMADH substrate have been determined and compared to one another as well as to the structure of the resting diferric MauG-preMADH complex. CO and NO each bind exclusively to the 5-coordinate high-spin heme with no change in ligation of the 6-coordinate low-spin heme. These structures reveal likely roles for amino acid residues in the distal pocket of the high-spin heme in oxygen binding and activation. Glu113 is implicated in the protonation of heme-bound diatomic oxygen intermediates in promoting cleavage of the O-O bond. Pro107 is shown to change conformation on the binding of each ligand and may play a steric role in oxygen activation by positioning the distal oxygen near Glu113. Gln103 is in a position to provide a hydrogen bond to the Fe(IV)O moiety that may account for the unusual stability of this species in MauG. [ABSTRACT FROM AUTHOR]

The diheme enzyme MauG catalyzes the post-translational modification of a precursor protein of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. This six-electron oxidation of preMADH requires long-range electron transfer (ET) as the structure of the MauG-preMADH complex reveals that the shortest distance between the modified residues of preMADH and the nearest heme of MauG is 14.0 Å [Jensen, L. M. R., Sanishvili, R., Davidson, V. L., and Wilmot, C. M. (2010) Science 327, 1392-1394). The kinetics of two ET reactions between MADH and MauG have been analyzed. Interprotein ET from quinol MADH to the high-valent bis- Fe(IV) form of MauG exhibits a Kd of 11.2 μM and a rate constant of 20 s-1. ET from diferrous MauG to oxidized TTQ of MADH exhibits a Kd of 10.1 μM and a rate constant of 0.07 s-1-1. These similar Kd values are much greater than that for the MauG-preMADH complex, indicating that the extent of VrQ maturity rather than its redox state influences complex formation. The difference in rate constants is consistent with a larger driving force for the faster reaction. Analysis of the structure of the MauG-preMADH complex in the context of ET theory and these results suggests that direct electron tunneling between the residues that form TTQ and the five-coordinate oxygen-binding heme is not possible, and that ET requires electron hopping via the six-coordinate heme. [ABSTRACT FROM AUTHOR]

MauG is a diheme enzyme that oxidizes two protein-bound tryptophan residues to generate a catalytic tryptophan tryptophylquinone cofactor within methyl- amine dehydrogenase. Upon the two-electron oxidation of bis-ferric MauG, the two c-type hemes exist as a spin- uncoupled bis-Fe(IV) species with only one binding oxygen, which is chemically equivalent to a single ferryl heme plus a sr porphyrin cation radical (Li, X. et al. (2008) Proc. NatI. Acad. Sci. U.S.A. 105, 8597-8600). The EPR spectrum of the nitrosyl complex of fully reduced MauG shows a single six-coordinate Fe(ll)-NO species, which is characteristic of a histidine-ligated Fe(ll)-NO moiety in the heme environment. Exposue of partially reduced MauG to NO reveals a redox equilibrium with facile electron transfer between hemes but with only one binding nitric oxide. Thus, the second heme is able to stabilize all three redox states of iron (Fe(Il), Fe(III), and Fe(IV)) in a six-coordinate protein-bound heme without binding exogenous ligands. This is unprecedented behavior for a protein-bound heme for which each of these redox states is relevant to the overall catalytic mechanism. The results also illustrate the electronic communication between the two iron centers, which function as a diheme unit rather than independent heme cofactors. [ABSTRACT FROM AUTHOR]

The diheme enzyme MauG catalyzes a six-electron oxidation required for posttranslational modification of a precursor of methylamine dehydrogenase (preMADH) to complete the biosynthesis of its protein-derived tryptophan tryptophylquinone (TTQ) cofactor. Crystallographic studies had shown that Pro107, which resides in the distal pocket of the high-spin heme of MauG, changes conformation upon binding of CO or NO to the heme iron. In this study, Pro 107 was converted to Cys, Val, and Ser by site-directed mutagenesis. The structures of each of these MauG mutant proteins in complex with preMADH were determined, as were their physical and catalytic properties. P107C MauG was inactive, and the crystal structure revealed that Cys107 had been oxidatively modified to a sulfinic acid. Mass spectrometry revealed that this modification was present prior to crystallization. P107V MauG exhibited spectroscopic and catalytic properties that were similar to those of wild-type MauG, but P107V MauG was more susceptible to oxidative damage. The P107S mutation caused a structural change that resulted in the five-coordinate high-spin heme being converted to a six-coordinate heme with a distal axial ligand provided by Glull3. EPR and resonance Raman spectroscopy revealed this heme remained high-spin but with gready increased rhombicity as compared to that of the axial signal of wild-type MauG. P107S MauG was resistant to reduction by dithionite and reaction with H2O2 and unable to catalyze TTQ biosynthesis. These results show that the presence of Pro107 is critical in maintaining the proper structure of the distal heme pocket of the high-spin heme of MauG, allowing exogenous ligands to bind and directing the reactivity of the heme-activated oxygen during catalysis, thus rninimizing the oxidation of other residues of MauG. [ABSTRACT FROM AUTHOR]

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 Kd for complex formation with MADH and altered the kinetic mechanism for the interprotein ET reaction. Values of reorganization energy (λ) and electronic coupling (HAB) 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 Kd, λ, and HAB 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. [ABSTRACT FROM AUTHOR]

The diheme enzyme MauG catalyzes the biosynthesis of tryptophan tryptophylquinone (TTQ), the protein-derived cofactor of methylamine dehydrogenase (MADH). This process requires the six-electron oxidation of a 119 kDa MADH precursor protein with incompletely synthesized TTQ (PreMADH). The kinetic mechanism of the initial two-electron oxidation of this natural substrate by MauG was characterized. The relative reactivity of free MauG toward H2O2 and the O2 analogue CO was essentially the same as that of MauG in the preformed enzyme-substrate complex. The addition of H2O2 to diferric MauG generated a diheme bis-Fe(IV) species [i.e., Fe(IV)=O/Fe(IV)] which formed at a rate of >300 s-1 and spontaneously returned to the diferric state at a rate of 2 × l0-4 s-1 in the absence of substrate. The reaction of bis-Fe(IV) MauG with PreMADH exhibited saturation behavior with a limiting first-order rate constant of 0.8 s-1 and a Kd of ≤ 1.5 μM for the MauG-PreMADH complex. The results were the same whether bis-Fe(IV) MauG was mixed with PreMADH or H2O2 was added to the preformed enzyme-substrate complex to generate bis-Fe(IV) MauG followed by reaction with PreMADH. Stopped-flow kinetic studies of the reaction of diferrous MauG with CO yielded a faster major transition with a bimolecular rate constant of 5.4 × 10-1 M-1 s-1, and slower transition with a rate of 16 s-1 which was independent of CO concentration. The same rates were obtained for binding of CO to diferrous MauG in complex with PreMADH. This demonstration of a random kinetic mechanism for the first two-electron oxidation reaction of MauG-dependent TTQ biosynthesis, in which the order of addition of oxidizing equivalent and substrate does not matter, is atypical of those of heme-dependent oxygenases that are not generally reactive toward oxygen in the absence of substrate. This kinetic mechanism is also distinct from that of the homologous diheme cytochrome c peroxidases that require a mixed valence state for activity. [ABSTRACT FROM AUTHOR]

Methylamine dehydrogenase (MADH) contains the protein-derived cofactor tryptophan tryptophyiquinone (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 α2β2 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 kcat of 0.20 ± 0.01 s-1 and Km of 6.6 ± 0.6 µM 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. [ABSTRACT FROM AUTHOR]

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 lCd for complex formation of M5 IA amicyanin was unchanged but the experimentally determined electronic coupling increased from 12 cm' 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 M5 IA mutation. These changes in ET parameters and rates for the reactions with M5 IA 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 M5 1K and M5 IL 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 M5IA 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 Met5 I 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. [ABSTRACT FROM AUTHOR]

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 ∼500 Ų of surface on each molecule, with one direct hydrogen bond linking them. The closest distance from TTQ to copper is 12.6 Å compared with a distance of 9.3 Å 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 ∼4.7 Å, the amicyanin bound to MADH appears to be rotated ∼90° 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. [ABSTRACT FROM AUTHOR]

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 (HAB) and reorganization energy (λ) 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 HAB increases from 12 to 78 cm-1, and λ 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 El 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 P520 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. [ABSTRACT FROM AUTHOR]

Quinohemoprotein amine dehydrogenase (QHNDH) of Paracoccus denitrificans contains a peptidyl quinone cofactor, cysteine tryptophylquinone, as well as intrapeptidyl thioether cross-links between Cys and Asp/Glu residues within the smallest γ-subunit of the αβγ heterotrimeric protein. A putative [Fe-S]-cluster-binding protein (ORF2 protein) encoded between the structural genes for the α- and -subunits of QHNDH in the n-butylamine-utilizing operon likely belongs to a Radical SAM (S-Ado-Met) superfamily that includes many proteins involved in vitamin biosynthesis and enzyme activation. In this study the role of ORF2 protein in the biogenesis of QHNDH has been explored. Although the wild-type strain of Paracoccus denitrificans produced an active, mature enzyme upon induction with n-butylamine, a mutant strain in which the ORF2 gene had been mostly deleted, neither grew in the n-butylamine medium nor showed QHNDH activity. When the mutant strain was transformed with an expression plasmid for the ORF2 protein, n-butylamine-dependent bacterial growth and QHNDH activity were restored. Site-specific mutations in the putative [Fe-S]-cluster or SAM binding motifs in the ORF2 protein failed to support bacterial growth. The α- and β-subunits were both detected in the periplasm of the mutant strain, whereas the γ-subunit polypeptide was accumulated in the cytoplasm and stained negatively for redox-cycling quinone staining. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis revealed that the γ-subunit isolated from the mutant strain had not undergone post-translational modification. These results unequivocally show that the putative [Fe-S]-cluster- and SAM-binding ORF2 protein is necessary for the posttranslational processing of γ-subunit, most likely participating in the formation of the intrapeptidyl thioether cross-links. [ABSTRACT FROM AUTHOR]

MauG is a novel 42 kDa diheme protein which is required for the biosynthesis of tryptophan tryptophyiquinone, the prosthetic group of methylamine dehydrogenase. The visible absorption and resonance Raman spectroscopic properties of each of the two c-type hemes and the overall redox properties of MauG are described. The absorption maxima for the Soret peaks of the oxidized and reduced hemes are 403 and 418 nm for the low-spin heme and 389 and 427 nm for the high-spin heme, respectively. The resonance Raman spectrum of oxidized MauG exhibits a set of marker bands at 1503 and 1588 cm-1 which exhibit frequencies similar to those of the v3 and v2 bands of c-type heme proteins with bis- histidine coordination. Another set of marker bands at 1478 and 1570 cm-1 is characteristic of a high-spin heme. Two distinct oxidation-reduction midpoint potential (Em) values of -159 and -244 mV are obtained from spectrochemical titration of MauG. However, the two v3 bands located at 1478 and 1503 cm-1 shift together to 1467 and 1492 cm-1, respectively, upon reduction, as do the Soret peaks of the low- and high-spin hemes in the absorption spectrum. Thus, the two hemes with distinct spectral properties are reduced and oxidized to approximately the same extent during redox titrations. This indicates that the high- and low-spin hemes have similar intrinsic Em values but exhibit negative redox cooperativity. After the first one-electron reduction of MauG, the electron equilibrates between hemes. This makes the second one-electron reduction of MauG more difficult. Thus, the two Em values do not describe redox properties of distinct hemes, but the first and second one-electron reductions of a diheme system with two equivalent hemes. The structural and mechanistic implications of these findings are discussed. [ABSTRACT FROM AUTHOR]

Quinohemoprotein amine dehydrogenase (QHNDH) possesses a cysteine tryptophylquinone (CTQ) prosthetic group that catalyzes the oxidative deamination of primary amines. In addition m CTQ, two heme c cofactors are present in QHNDH that mediate the transfer of the substrate-derived electrons from CTQ re an external electron acceptor. Steady-state kinetic assays yielded relatively small k[sub cat] values (<6 s[sup -1]), and the rate-limiting step appears re be the interprotein electron transfer from heme in QHNDH to the external electron acceptor. Transient kinetic studies of the CTQ-dependent reduction of heme in QHNDH by amine substrates yielded different rate constants for different substrates (72, 190, and 162 s[sup -1] for methylamine, butylamine, and benzylamine, respectively). Deuterium kinetic isotope effect (KIE) values of 5.3, 3.9, and 8.5 were observed, respectively, for the reactions of methylamine, butylamine, and benzylamine. These results suggest that the abstraction of a proton from the α-methylene group of the substrate, which occurs concomitant with CTQ reduction, is the rate-limiting step in the CTQ-dependent reduction of hemes in QHNDH by these amine substrates. In contrast, the reaction of 2-phenylethylamine with QHNDH does not exhibit a significant KIE ([sup H]k[sub 3]/[sup D]k[sub 3] = 1.05) and exhibits a much smaller rate constant of 16 s[sup -1]. This suggests that for 2-phenylethylamine, the rate-limiting step in the single-turnover reaction ts either hydrolysis of the imine reaction intermediate from CTQ or product release prior m intraprotein electron transfer Analysis of the products of the reactions of QHNDH with chiral deuterated 2-phenylethylamines demonstrated that the enzyme abstracts the pro-S proton of the substrate in a highly stereospecific manner. Inspection of the crystal structure of phenylhydrazine-inhibited QHNDH suggests that Asp33γ is the residue that performs the proton abstraction. [ABSTRACT FROM AUTHOR]

The biosynthesis of methylamine dehydrogenase (MADH) from Paracoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits. None of these gene products have been previously isolated. One of these, mauG, exhibits sequence similarity to diheme cytochrome c peroxidases and is required for the synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group of MADH. A system was developed for the homologous expression of MauG in P. denitrificans. Its signal sequence was correctly processed, and it was purified from the periplasmic cell fraction. The protein contains two covalent c-type hemes, as predicted from the deduced sequence. EPR spectroscopy reveals that the protein as isolated possesses about equal amounts of one high-spin heme with axial symmetry and one low-spin heme with rhombic symmetry. The low-spin heme contains a major and minor component suggesting a small degree of heme heterogeneity. The high-spin heme and the major low-spin heme component each exhibit resonances that are atypical of c-type hemes and dissimilar to those reported for diheme cytochrome c peroxidases. MauG exhibited only very weak peroxidase activity when assayed with either c-type cytochromes or o-dianisidine as an electron donor. Fully reduced MauG was shown to bind carbon monoxide and could be reoxidized by oxygen. The relevance of these unusual properties of MauG is discussed in the context of its role in TTQ biogenesis. [ABSTRACT FROM AUTHOR]

Cofactors made from constitutive amino acids in proteins are now known to be relatively common. A number of these involve the generation of quinone cofactors, such as topaquinone in the copper-containing amine oxidases, and lysine tyrosylquinone in lysyl oxidase. The biogenesis of the quinone cofactor tryptophan tryptophylquinone (TTQ) in methylamine dehydrogenase (MADH) involves the posttranslational modification of two constitutive Trp residues (Trp[sup β57] and Trp[sup β108] in Paracoccus denitrificans MADH). The modifications for generating TTQ are the addition of two oxygens to the indole ring of Trp[sup β57] and the formation of a covalent cross-link between C∈3 of Trp[sup β57] and Cδ1 of Trp[sup β108]. The order in which these events occur is unknown. To investigate the role Trp[sup β108] may play in this process, this residue was mutated to both a His (βW108H) and a Cys (βW108C) residue. For each mutant, the majority of the protein that was isolated was inactive and exhibited weaker subunit-subunit interactions than native MADH. Analysis by mass spectrometry suggested that the inactive protein was a biosynthetic intermediate with only one oxygen atom incorporated into Trp[sup β57] and no cross-link with residue β108. However, in each mutant preparation, a small percentage of the mutant enzyme was active and appears to possess a functional tryptophylquinone cofactor. In the case of βW108C, this cofactor may be identical to cysteine tryptophylquinone, recently described in the bacterial quinohemoprotein amine dehydrogenase. In βW 108H, the active cofactor is presumably a histidine tryptophylquinone, which has not been previously described, and represents the synthesis of a novel quinone protein cofactor. [ABSTRACT FROM AUTHOR]

Methylamine dehydrogenase (MADH) possesses an α[sub 2]/β[sub 2] structure with each smaller β subunit possessing a tryptophan tryptophylquinone (TTQ) prosthetic group. Phe55 of the α subunit is located where the substrate channel from the enzyme surface opens into the active site. Site-directed mutagenesis of αPhe55 has revealed roles for this residue in determining substrate specificity and binding monovalent cations at the active site. It is now shown that the αF55A 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[sup -1]. The crystal structure of αF55A MADH in complex with its electron acceptors, amicyanin and cytochrome c-55 li, 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 αF55A 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. [ABSTRACT FROM AUTHOR]

Describes the improvement of sensitivity of histamine sensor through engineered methylamine dehydrogenase. Biological component of enzyme-based electrodes; Determination the specificity of methylamine dehydrogenase; Conversion of phenylalanine into alanine.

The article presents a study which confirmed that methylamine dehydrogenase contains only one exchangeable carbonyl consistent with the crystal structure in which the C6 carbonyl is exposed to solvent while the C7 carbonyl is hydrogen-bonded to a backbone amide. Any oxygen atom added to the C6 position of tryptophan tryptophylquinone during synthesis will exchange with solvent upon cofactor maturation.

The article reports that tryptophan tryptophylquinone (TTQ) is the prosthetic group of methylamine dehydrogenase (MADH) and aromatic amine dehydrogenase. It is synthesized through post-translational modification of two endogenous tryptophan residues. This modification involves two oxygenation reactions and one cross-linking reaction. The genes that encode the MADH subunits, together with nine other genes that relate to MADH expression and function, are clustered in the methylamine utilization (mau) locus. Four genes, mauFEDG, were shown to be essential for MADH maturation.

Describes the direct detection of [sup 15]N nuclear magnetic resonance of the tryptophan tryptophylquinone aminoquinol reaction intermediate of methylamine dehydrogenase. Catalysis of the transformations of biological amines; Factors influencing the oxidative deamination of primary amines to the corresponding aldehyde, ammonium, and two electrons; Reoxidation of aminoquinol.