A joint x-ray and neutron study on amicyanin reveals the role of protein dynamics in electron transfer.

The joint x-ray/neutron diffraction model of the Type I copper protein, amicyanin from Paracoccus denitrificans was determined at 1.8 A resolution. The protein was crystallized using reagents prepared in D(2)O. About 86% of the amide hydrogen atoms are either partially or fully exchanged, which correlates well with the atomic depth of the amide nitrogen atom and the secondary structure type, but with notable exceptions. Each of the four residues that provide copper ligands is partially deuterated. The model reveals the dynamic nature of the protein, especially around the copper-binding site. A detailed analysis of the presence of deuterated water molecules near the exchange sites indicates that amide hydrogen exchange is primarily due to the flexibility of the protein. Analysis of the electron transfer path through the protein shows that residues in that region are highly dynamic, as judged by hydrogen/deuterium exchange. This could increase the rate of electron transfer by transiently shortening through-space jumps in pathways or by increasing the atomic packing density. Analysis of C-HX bonding reveals previously undefined roles of these relatively weak H bonds, which, when present in sufficient number can collectively influence the structure, redox, and electron transfer properties of amicyanin.

A preliminary time-of-flight neutron diffraction study on amicyanin from Paracoccus denitrificans.

Crystals of the blue copper protein amicyanin suitable for neutron diffraction were grown by the sitting-drop method, followed by repeated macroseeding using solutions prepared with D(2)O. Although the crystal sizes were the same, crystals grown using solutions made up in H(2)O in the initial stages of macroseeding and solutions with D(2)O in later stages did not diffract neutrons well. However, when the protein was initially exchanged with buffered D(2)O and then crystallized and also macroseeded using solutions made up in D(2)O throughout, the crystals diffracted neutrons to high resolution. One of those crystals was used to collect a data set to a resolution of 1.9 A.

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.

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.

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.

Molecular basis for complex formation between methylamine dehydrogenase and amicyanin revealed by inverse mutagenesis of an interprotein salt bridge.

Methylamine dehydrogenase (MADH) and amicyanin form a physiologic complex which is required for interprotein electron transfer. The crystal structure of this protein complex is known, and the importance of certain residues on amicyanin in its interaction with MADH has been demonstrated by site-directed mutagenesis. In this study, site-directed mutagenesis of MADH, kinetic data, and thermodynamic analysis are used to probe the molecular basis for stabilization of the protein complex by an interprotein salt bridge between Arg99 of amicyanin and Asp180 of the alpha subunit of MADH. This paper reports the first site-directed mutagenesis of MADH, as well as the construction, heterologous expression, and characterization of a six-His-tagged MADH. alpha Asp180 of MADH was converted to arginine to examine the effect on complex formation with native and mutant amicyanins. This mutation had no effect on the parameters for methylamine oxidation by MADH, but significantly affected its interaction with amicyanin. Of the native and mutant proteins that were studied, their observed order of affinity for each other was as follows: native MADH and native amicyanin > native MADH and R99D amicyanin > alpha D180R MADH and native amicyanin > alpha D180R MADH and R99D amicyanin, and alpha D180R MADH and R99L amicyanin. The alpha D180R mutation also eliminated the ionic strength dependence of the reaction of MADH with amicyanin that is observed with wild-type MADH. Interestingly, the inverse mutation pair of alpha D180R MADH and R99D amicyanin did not restore the favorable salt bridge, but instead disrupted complex formation much more severely than did either individual mutation. These results are explained using molecular modeling and thermodynamic analysis of the kinetic data to correlate the energy contributions of specific stabilizing and destabilizing interactions that are present in the wild-type and mutant complexes. A model is also proposed to describe the sequence of events that leads to stable complex formation between MADH and amicyanin.

Amperometric detection of histamine with a methylamine dehydrogenase polypyrrole-based sensor.

Methylamine dehydrogenase (MADH) has been immobilized in a polpyrrole (PPy) film on an electrode surface and used as an amine sensor for the determination of primary amines. Its response to histamine has been characterized in detail. The PPy film containing MADH was formed electrochemically on a gold minielectrode (1-mm diameter) in the presence of ferricyanide. The film was then coated with Nafion. This enzyme electrode did not require any additional cofactors and was not sensitive to oxygen. It exhibited a maximum response current to histamine at applied potentials of 0.24-0.33 V and at pH 7.5-8.5. This MADH-PPy sensor exhibited a response time of less than 3 s. The immobilized MADH on the electrode exhibited Michaelis-Menten behavior similar to that of the free enzyme in solution with a Km value of 1.3 mM. This sensor could be used to reliably detect histamine over a concentration range from approximately 25 microM to 4 mM. This is the first example of a biosensor that uses an immobilized enzyme that possesses the tryptophan tryptophylquinone prosthetic group.

Effects of kinetic coupling on experimentally determined electron transfer parameters.

Coupled electron transfer (ET) occurs when a relatively slow nonadiabatic ET reaction is preceded by a rapid but unfavorable adiabatic reaction that is required to activate the system for ET. As a consequence of this, the observed ET rate constant (k(ET)) is an apparent value equal to the product of the true k(ET) and the equilibrium constant for the preceding reaction step. Analysis of such reactions by ET theory may yield erroneous values for the reorganizational energy (lambda), electronic coupling (H(AB)), and ET distance that are associated with the true k(ET). If the DeltaG degrees dependence of the rate of a coupled ET reaction is analyzed, an accurate value of lambda will be obtained but the experimentally determined H(AB) will be less than the true H(AB) and the ET distance will be greater than the true distance. If the temperature dependence of the rate of a coupled ET reaction is analyzed, the experimentally determined value of lambda will be greater than the true lambda. The magnitude of this apparent lambda will depend on the magnitude of DeltaH degrees for the unfavorable reaction step that precedes ET. The experimentally determined values of H(AB) and distance will be accurate if DeltaS degrees for the preceding reaction is zero. If DeltaS degrees is positive, then H(AB) will be greater than the true value and the distance will be less than the true value. If DeltaS degrees is negative, then H(AB) will be less than the true value and the distance will be greater than the true value. Data sets for coupled ET reactions have been simulated and analyzed by ET theory to illustrate these points.

What controls the rates of interprotein electron-transfer reactions.

Rates of electron-transfer (ET) reactions are dependent on driving force, reorganizational energy, distance, and the nature of the medium which the electron must traverse. In kinetically complex biological systems, non-ET reactions may be required to activate the system for ET and may also influence the observed rates. Studies of ET from tryptophan tryptophylquinone to copper to heme in the methylamine dehydrogenase-amicyanin-cytochrome c-551i ET complex, as well as studies of other physiologic redox protein complexes, are used to illustrate the combination of factors which control rates of interprotein ET reactions.

Tyr(30) of amicyanin is not critical for electron transfer to cytochrome c-551i: implications for predicting electron transfer pathways.

A Pathways analysis of the methylamine dehydrogenase-amicyanin-cytochrome c-551i protein electron transfer (ET) complex predicts two sets of ET pathways of comparable efficiency from the type I copper of amicyanin to the heme of cytochrome c-551i. In one pathway, the electron exits copper via the Cys(92) copper ligand, and in the other, it exits via the Met(98) copper ligand. If the Pathways algorithm is modified to include contributions from the anisotropy of metal-ligand coupling, independent of differences in copper-ligand bond length, then the pathways via Cys(92) are predicted to be at least 100-fold more strongly coupled than the pathways via any of the other copper ligands. All of the favored pathways via Cys(92) include a through-space jump from Cys(92) to the side chain of Tyr(30). To determine whether or not the pathways via Cys(92) are preferentially used for ET, Tyr(30) was changed to other amino acid residues by site-directed mutagenesis. Some mutant proteins were very unstable suggesting a role for Tyr(30) in stabilizing the protein structure. Y30F and Y30I mutant amicyanins could be isolated and analyzed. For the Y30I mutant, the modified Pathways analysis which favors ET via Cys(92) predicts a decrease in ET rate of at least two orders of magnitude, whereas the standard Pathways analysis predicts no change in ET rate since ET via Met(98) is not affected. Experimentally, the ET rates of the Y30I and Y30F mutants were indistinguishable from that of wild-type amicyanin. Likely explanations for these observations are discussed as are their implications for predicting pathways for ET reactions of metalloproteins.

Gated and ungated electron transfer reactions from aromatic amine dehydrogenase to azurin.

Interprotein electron transfer (ET) occurs between the tryptophan tryptophylquinone (TTQ) prosthetic group of aromatic amine dehydrogenase (AADH) and copper of azurin. The ET reactions from two chemically distinct reduced forms of TTQ were studied: an O-quinol form that was generated by reduction by dithionite, and an N-quinol form that was generated by reduction by substrate. It was previously shown that on reduction by substrate, an amino group displaces a carbonyl oxygen on TTQ, and that this significantly alters the rate of its oxidation by azurin (Hyun, Y-L., and Davidson V. L. (1995) Biochemistry 34, 12249-12254). To determine the basis for this change in reactivity, comparative kinetic and thermodynamic analyses of the ET reactions from the O-quinol and N-quinol forms of TTQ in AADH to the copper of azurin were performed. The reaction of the O-quinol exhibited values of electronic coupling (H(AB)) of 0.13 cm(-1) and reorganizational energy (lambda) of 1.6 eV, and predicted an ET distance of approximately 15 A. These results are consistent with the ET event being the rate-determining step for the redox reaction. Analysis of the reaction of the N-quinol by Marcus theory yielded an H(AB) which exceeded the nonadiabatic limit and predicted a negative ET distance. These results are diagnostic of a gated ET reaction. Solvent deuterium kinetic isotope effects of 1.5 and 3.2 were obtained, respectively, for the ET reactions from O-quinol and N-quinol AADH indicating that transfer of an exchangeable proton was involved in the rate-limiting reaction step which gates ET from the N-quinol, but not the O-quinol. These results are compared with those for the ET reactions from another TTQ enzyme, methylamine dehydrogenase, to amicyanin. The mechanism by which the ET reaction of the N-quinol is gated is also related to mechanisms of other gated interprotein ET reactions.

Localization of periplasmic redox proteins of Alcaligenes faecalis by a modified general method for fractionating gram-negative bacteria.

A lysozyme-osmotic shock method is described for fractionation of Alcaligenes faecalis which uses glucose to adjust osmotic strength and multiple osmotic shocks. During phenylethylamine-dependent growth, aromatic amine dehydrogenase, azurin, and a single cytochrome c were localized in the periplasm. Their induction patterns are different from those for the related quinoprotein methylamine dehydrogenase and its associated redox proteins.

Heterologous expression of correctly assembled methylamine dehydrogenase in Rhodobacter sphaeroides.

The biosynthesis of methylamine dehydrogenase (MADH) from Paracoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits, mauB and mauA. The accessory gene products appear to be required for proper export of the protein to the periplasm, synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group, and formation of several structural disulfide bonds. To accomplish the heterologous expression of correctly assembled MADH, eight genes from the methylamine utilization gene cluster of P. denitrificans, mauFBEDACJG, were placed under the regulatory control of the coxII promoter of Rhodobacter sphaeroides and introduced into R. sphaeroides by using a broad-host-range vector. The heterologous expression of MADH was constitutive with respect to carbon source, whereas the native mau promoter allows induction only when cells are grown in the presence of methylamine as a sole carbon source and is repressed by other carbon sources. The recombinant MADH was localized exclusively in the periplasm, and its physical, spectroscopic, kinetic and redox properties were indistinguishable from those of the enzyme isolated from P. denitrificans. These results indicate that mauM and mauN are not required for MADH or TTQ biosynthesis and that mauFBEDACJG are sufficient for TTQ biosynthesis, since R. sphaeroides cannot synthesize TTQ. A similar construct introduced into Escherichia coli did not produce detectable MADH activity or accumulation of the mauB and mauA gene products but did lead to synthesizes of amicyanin, the mauC gene product. This finding suggests that active recombinant MADH is not expressed in E. coli because one of the accessory gene products is not functionally expressed. This study illustrates the potential utility of R. sphaeroides and the coxII promoter for heterologous expression of complex enzymes such as MADH which cannot be expressed in E. coli. These results also provide the foundation for future studies on the molecular mechanisms of MADH and TTQ biosynthesis, as well as a system for performing site-directed mutagenesis of the MADH gene and other mau genes.

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.

Structure, function, and applications of tryptophan tryptophylquinone enzymes.

Tryptophan and tyrosine residues in proteins may be posttranslationally modified to form enzyme cofactors. Tryptophan tryptophylquinone (TTQ), the cofactor of methylamine dehydrogenase (MADH), is formed by covalent cross-linking of two tryptophan residues and incorporation of two oxygen atoms into one of the indole rings to form a quinone. MADH converts primary amines to their corresponding aldehydes plus ammonia. During the catalytic cycle, TTQ mediates electron transfer from substrate to a copper protein, amicyanin. These electrons are transferred to the respiratory chain via a c-type cytochrome. Structural, kinetic and site-directed mutagenesis studies have characterized protein-protein interactions, and mechanisms of catalysis and electron transfer by TTQ. Preliminary results obtained with MADH enzyme-electrodes demonstrate the potential for quinoprotein-based biosensors.

Molecular basis for interprotein complex-dependent effects on the redox properties of amicyanin.

The quinoprotein methylamine dehydrogenase (MADH), type I copper protein amicyanin, and cytochrome c-551i form a complex within which interprotein electron transfer occurs. It was known that complex formation significantly lowered the oxidation-reduction midpoint potential (Em) value of amicyanin, which facilitated an otherwise thermodynamically unfavorable electron transfer to cytochrome c-551i. Structural, mutagenesis, and potentiometric studies have elucidated the basis for this complex-dependent change in redox properties. Positively charged amino acid residues on the surface of amicyanin are known to stabilize complex formation with MADH and influence the ionic strength dependence of complex formation via electrostatic interactions. Altering the charges of these residues by site-directed mutagenesis had no effect on the Em value of amicyanin, ruling out charge neutralization as the basis for the complex-dependent changes in redox properties. The Em value of free amicyanin varies with pH and exhibits a pKa value for the reduced form of 7.5. The crystal structure of reduced amicyanin at pH 4.4 reveals that His95, which serves as a ligand for Cu2+, has rotated by 180 degrees about the Cbeta-Cgamma bond relative to its position in oxidized amicyanin and is no longer in the copper coordination sphere. At pH 7.7, the crystal structure of reduced amicyanin contains an approximately equal distribution of two active-site conformers. One is very similar to the structure of reduced amicyanin at pH 4.4, and the other is very similar to the structure of oxidized amicyanin at pH 4.8. Potentiometric analysis of amicyanin in complex with MADH indicates that its Em value is not pH-dependent from pH 6.5 to 8.5, and exhibits an Em value similar to that of free amicyanin at high pH. The structure of reduced amicyanin at pH 4.4, with His95 protonated and "flipped", was modeled into the structure of the complex of oxidized amicyanin with MADH. This showed that in the complex, the redox-linked pH-dependent rotation of His95 is hindered because it would cause an overlap of van der Waals' radii with residues of MADH. These results demonstrate that protein-protein interactions profoundly affect the redox properties of this type I copper protein by restricting a pH-dependent, redox-linked conformational change of one of the copper ligands.

Electron transfer from the aminosemiquinone reaction intermediate of methylamine dehydrogenase to amicyanin.

The tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase (MADH) is covalently modified by substrate-derived nitrogen during its two-electron reduction by methylamine to form an aminoquinol (N-quinol). An N-semiquinone, which retains the substrate-derived N, is the intermediate during the two sequential one-electron oxidations of N-quinol MADH by its physiologic electron acceptor, amicyanin. Electron transfer (ET) from N-quinol MADH to amicyanin is gated by the deprotonation of the substrate-derived amino group on TTQ in the enzyme active site, whereas ET reactions from dithionite-reduced quinol and semiquinone forms of MADH are rate-limited by the ET event. The ET reaction from the N-semiquinone intermediate is shown not to be gated, but rate-limited by the ET step. Marcus analysis of the reaction reveals that the ET reaction from the N-semiquinone MADH to amicyanin exhibits the same reorganizational energy and electronic coupling as do the ET reactions of the dithionite-reduced O-quinol and O-semiquinone forms. The rates of the ET reactions of these three different redox forms of MADH exhibit a DeltaG degrees dependence which is predicted by Marcus theory. The ET reaction of the N-semiquinone is relatively insensitive to pH and salt, and does not exhibit a primary kinetic solvent isotope effect over the range of pH and cation concentrations studied. These properties are similar to those of the ET reaction of quinol MADH and different from those of the gated reaction of N-quinol MADH, whose rate varies considerably with pH and concentrations of specific monovalent cations. Thus, the covalent incorporation of substrate-derived N into TTQ is not alone sufficient to cause gating of ET. It affects the rate and DeltaG degrees for the ET reaction from the TTQ semiquinone by altering its redox potential, but it does not alter the reorganizational energy and electronic coupling associated with ET from TTQ to amicyanin.