Aromatic Amine Dehydrogenase Research Articles

Ab Initio QM/MM Modeling of the Rate-Limiting Proton Transfer Step in the Deamination of Tryptamine by Aromatic Amine Dehydrogenase.

Aromatic amine dehydrogenase (AADH) and related enzymes are at the heart of debates on the roles of quantum tunneling and protein dynamics in catalysis. The reaction of tryptamine in AADH involves significant quantum tunneling in the rate-limiting proton transfer step, shown by large H/D primary kinetic isotope effects (KIEs), with unusual temperature dependence. We apply correlated ab initio combined quantum mechanics/molecular mechanics (QM/MM) methods, at levels up to local coupled cluster theory (LCCSD(T)/(aug)-cc-pVTZ), to calculate accurate potential energy surfaces for this reaction, which are necessary for quantitative analysis of tunneling contributions and reaction dynamics. Different levels of QM/MM treatment are tested. Multiple pathways are calculated with fully flexible transition state optimization by the climbing-image nudged elastic band method at the density functional QM/MM level. The average LCCSD(T) potential energy barriers to proton transfer are 16.7 and 14.0 kcal/mol for proton transfer to the two carboxylate atoms of the catalytic base, Asp128β. The results show that two similar, but distinct pathways are energetically accessible. These two pathways have different barriers, exothermicity and curvature, and should be considered in analyses of the temperature dependence of reaction and KIEs in AADH and other enzymes. These results provide a benchmark for this prototypical enzyme reaction and will be useful for developing empirical models, and analyzing experimental data, to distinguish between different conceptual models of enzyme catalysis.

Incorporating Fast Protein Dynamics into Enzyme Design: A Proposed Mutant Aromatic Amine Dehydrogenase

In recent years, there has been encouraging progress in the engineering of enzymes that are designed to catalyze reactions not accelerated by natural enzymes. We tested the possibility of reengineering an existing enzyme by introducing a fast protein motion that couples to the reaction. Aromatic amine dehydrogenase is a system that has been shown to use a fast substrate motion as part of the reaction mechanism. We identified a mutation that preserves this fast motion but also introduces a favorable fast motion near the active site that did not exist in the native enzyme. Transition path sampling was used for the analysis of the atomic details of the mechanism.

Examining the importance of dynamics, barrier compression and hydrogen tunnelling in enzyme catalysed reactions

Nuclear quantum mechanical tunnelling is important in enzyme-catalysed H-transfer reactions. This viewpoint has arisen after a number of experimental studies have described enzymatic reactions with kinetic isotope effects that are significantly larger than the semiclassical limit. Other experimental evidence for tunnelling, and the potential role of promoting vibrations that transiently compress the reaction barrier, is more indirect, being derived from the interpretation of e.g. mutational analyses of enzyme systems and temperature perturbation studies of reaction rates/kinetic isotope effects. Computational simulations have, in some cases, determined exalted kinetic isotope effects and tunnelling contributions, and identified putative promoting vibrations. In this review, we present the available evidence – both experimental and computational – for environmentally-coupled Htunnelling in several enzyme systems, namely aromatic amine dehydrogenase and members of the Old Yellow Enzyme family. We then consider the relative importance of tunnelling contributions to these reactions. We find that the tunnelling contribution to these reactions confers a rate enhancement of ∼1000-fold. Without tunnelling, a 1000-fold reduction in activity would seriously impair cellular metabolism. We therefore infer that tunnelling is crucial to host organism viability thereby emphasising the general importance of tunnelling in biology.

Tunneling in enzymatic and nonenzymatic hydrogen transfer reactions

AbstractThis paper is a response to an invitation to share my viewpoint by writing an opinion piece (not a review) on proton tunneling, especially from the point of view of whether it has a greater importance in enzymatic reactions than in other reactions. The paper begins with a discussion of the emergence of a conceptual framework for including tunneling in reaction rate calculations; the framework is general enough to include not only transfer of protons but also transfer of hydrogen atoms and hydride ions and their isotopes, and not only enzymatically catalyzed reactions but also nonenzymatic reactions. Then the paper discusses the special issues that arise when the reaction rate under consideration is for an enzyme‐catalyzed reaction. The emphasis is on physical considerations in reaction rate calculations, not on system‐specific comparison of results for different modes of reaction. It is argued that enzymatic and nonenzymatic reactions may be treated within the same basic framework except that ensemble averaging, which is not usually required for gas‐phase reactions, is essential for treating enzyme reactions. Enzymes explicitly discussed include methylamine dehydrogenase, aromatic amine dehydrogenase, E. coli dihydrofolate reductase, hyperthermophilic dihydrofolate reductase, liver alcohol dehydrogenase, methylmalonyl‐CoA mutase, soybean lipoxygenase, copper amine oxidase, pentaerythritol tetranitrate reductase, morphinone reductase, enolase, xylose isomerase, and 4‐oxalocrotonate tautomerase. Copyright © 2010 John Wiley & Sons, Ltd.

Optimal control design of laser pulses for mode specific vibrational excitation in an enzyme–substrate complex

High level combined quantum mechanics/molecular mechanics (QM/MM) methods are used to calculate the potential energy and dipole moment surfaces for the motion of the labile proton in an intermediate in the catalytic cycle of aromatic amine dehydrogenase (AADH). Quantum tunnelling is important in transfer of this tryptamine proton. Optimal control theory is used here to design laser pulses to excite the labile proton from its lowest to selected vibrationally excited states. Such pulses leading to excitation of specific vibrational modes might in future be useful to promote reactivity by enhancing proton tunnelling; we discuss practical difficulties in carrying this out.

Computer simulations of quantum tunnelling in enzyme-catalysed hydrogen transfer reactions

Transfer of hydrogen as a proton, hydride or hydrogen atom is an important step in many enzymic reactions. Experiments show kinetic isotope effects (KIEs) for some enzyme-catalysed hydrogen transfer reactions that deviate significantly from the limits imposed by considering the differences in mass of the isotopes alone (i.e. the semiclassical limit). These KIEs can be explained if the transfer of the hydrogen species occurs via a quantum mechanical tunnelling mechanism. The unusual temperature dependence of some KIEs has led to suggestions that enzymes have evolved to promote tunnelling through dynamics - a highly controversial hypothesis. Molecular simulations have a vital role in resolving these questions, providing a level of detail of analysis not possible through experiments alone. Here, we review computational molecular modelling studies of quantum tunnelling in enzymes, in particular focusing on the enzymes soybean lipoxygenase-1 (SLO-1), dihydrofolate reductase (DHFR), methylamine dehydrogenase (MADH) and aromatic amine dehydrogenase (AADH) to illustrate the current controversy regarding the importance of quantum effects in enzyme catalysis.

New insights into the multi-step reaction pathway of the reductive half-reaction catalysed by aromatic amine dehydrogenase: a QM/MM study

Computational insight into the multi-step reaction cycle of aromatic amine dehydrogenase is presented, identifying the energy landscape and pathway for multiple proton transfers. This atomistic picture of the reaction sequence--including short-lived reaction intermediates and a stepwise reaction mechanism--bridges the gap between a small number of crystallographic snapshots.

Barrier Compression and Its Contribution to Both Classical and Quantum Mechanical Aspects of Enzyme Catalysis

It is generally accepted that enzymes catalyze reactions by lowering the apparent activation energy by transition state stabilization or through destabilization of ground states. A more controversial proposal is that enzymes can also accelerate reactions through barrier compression—an idea that has emerged from studies of H-tunneling reactions in enzyme systems. The effects of barrier compression on classical (over-the-barrier) reactions, and the partitioning between tunneling and classical reaction paths, have largely been ignored. We performed theoretical and computational studies on the effects of barrier compression on the shape of potential energy surfaces/reaction barriers for model (malonaldehyde and methane/methyl radical anion) and enzymatic (aromatic amine dehydrogenase) proton transfer systems. In all cases, we find that barrier compression is associated with an approximately linear decrease in the activation energy. For partially nonadiabatic proton transfers, we show that barrier compression enhances, to similar extents, the rate of classical and proton tunneling reactions. Our analysis suggests that barrier compression—through fast promoting vibrations, or other means—could be a general mechanism for enhancing the rate of not only tunneling, but also classical, proton transfers in enzyme catalysis.

Assignment of the Vibrational Spectra of Enzyme-Bound Tryptophan Tryptophyl Quinones Using a Combined QM/MM Approach

Fourier transform infrared (FTIR) spectroscopy can be used to provide a detailed time-resolved probe of reaction intermediates in enzyme-catalyzed systems. Accurate assignment of the respective chemical species being studied is key to the success of this approach. The plethora of signals from the protein environment, leading to complexity in the spectra, presents a particular challenge. Here we present a combined QM/MM-based approach that can be used to assign key resonances in the FTIR spectrum of tryptophan tryptophyl quinone (TTQ) in the TTQ-dependent quinoprotein aromatic amine dehydrogenase (AADH). We show that consideration of the cofactor alone is not sufficient to identify correctly the experimentally observed resonances-inclusion of the protein is required for this. However, to enable accurate peak assignment, a stepwise approach is needed that builds up increasing levels of complexity from a simple system. This study serves as a benchmark for future QM/MM-based studies to predict the spectroscopic changes during the interconversion of intermediates in the reductive half-reaction catalyzed by AADH, and more generally for using a combined QM/MM approach to calculate spectroscopic data of protein cofactors and cofactor-based adducts.

The role of reaction energy and hydrogen bonding in the reaction path of enzymatic proton transfers

AbstractThe reaction path of the Interacting‐State Model is used with the Transition‐State Theory and the semiclassical correction for tunneling (ISM/scTST) to calculate proton transfer rates in rate‐determining CH bond breaking by enzymes such as mandelate racemase, triose‐phosphate isomerase, methylamine dehydrogenase (MADH), and aromatic amine dehydrogenase (AADH), as well as of the analogous uncatalyzed proton transfers in aqueous solution. This method employs the reaction energy, the bond distances in reactants and products, and properties of the reactive bonds to calculate the proton transfer rates. The comparison between the molecular factors that control reactivity in solution and in the enzymes reveals the following salient features of enzyme catalysis: (1) the change from a bimolecular reaction in solution to an intramolecular reaction with negligible activation entropy in the enzyme; (2) energetic stabilization of the intermediates; and (3) extreme tunneling corrections when hydrogen bonding to a carbon atom is present in the reaction path. Copyright © 2008 John Wiley & Sons, Ltd.

Driving Force Analysis of Proton Tunnelling Across a Reactivity Series for an Enzyme‐Substrate Complex

Quantitative structure-activity relationships are widely used to probe C-H bond breakage by quinoprotein enzymes. However, we showed recently that p-substituted benzylamines are poor reactivity probes for the quinoprotein aromatic amine dehydrogenase (AADH) because of a requirement for structural change in the enzyme-substrate complex prior to C-H bond breakage. This rearrangement is partially rate limiting, which leads to deflated kinetic isotope effects for p-substituted benzylamines. Here we report reactivity (driving force) studies of AADH with p-substituted phenylethylamines for which the kinetic isotope effect (approximately 16) accompanying C-H/C-(2)H bond breakage is elevated above the semi-classical limit. We show bond breakage occurs by quantum tunnelling and that within the context of the environmentally coupled framework for H-tunnelling the presence of the p-substituent places greater demand on the apparent need for fast promoting motions. The crystal structure of AADH soaked with phenylethylamine or methoxyphenylethylamine indicates that the structural change identified with p-substituted benzylamines should not limit the reaction with p-substituted phenylethylamines. This is consistent with the elevated kinetic isotope effects measured with p-substituted phenylethylamines. We find a good correlation in the rate constant for proton transfer with bond dissociation energy for the reactive C-H bond, consistent with a rate that is limited by a Marcus-like tunnelling mechanism. As the driving force becomes larger, the rate of proton transfer increases while the Marcus activation energy becomes smaller. This is the first experimental report of the driving force perturbation of H-tunnelling in enzymes using a series of related substrates. Our study provides further support for proton tunnelling in AADH.

The enzyme aromatic amine dehydrogenase induces a substrate conformation crucial for promoting vibration that significantly reduces the effective potential energy barrier to proton transfer

The role of promoting vibrations in enzymic reactions involving hydrogen tunnelling is contentious. While models incorporating such promoting vibrations have successfully reproduced and explained experimental observations, it has also been argued that such vibrations are not part of the catalytic effect. In this study, we have employed combined quantum mechanical/molecular mechanical methods with molecular dynamics and potential energy surface calculations to investigate how enzyme and substrate motion affects the energy barrier to proton transfer for the rate-limiting H-transfer step in aromatic amine dehydrogenase (AADH) with tryptamine as substrate. In particular, the conformation of the iminoquinone adduct induced by AADH was found to be essential for a promoting vibration identified previously-this lowers significantly the 'effective' potential energy barrier, that is the barrier which remains to be surmounted following collective, thermally equilibrated motion attaining a quantum degenerate state of reactants and products. When the substrate adopts a conformation similar to that in the free iminoquinone, this barrier was found to increase markedly. This is consistent with AADH facilitating the H-transfer event by holding the substrate in a conformation that induces a promoting vibration.

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.

Analysis of Classical and Quantum Paths for Deprotonation of Methylamine by Methylamine Dehydrogenase

The hydrogen-transfer reaction catalysed by methylamine dehydrogenase (MADH) with methylamine (MA) as substrate is a good model system for studies of proton tunnelling in enzyme reactions--an area of great current interest--for which atomistic simulations will be vital. Here, we present a detailed analysis of the key deprotonation step of the MADH/MA reaction and compare the results with experimental observations. Moreover, we compare this reaction with the related aromatic amine dehydrogenase (AADH) reaction with tryptamine, recently studied by us, and identify possible causes for the differences observed in the measured kinetic isotope effects (KIEs) of the two systems. We have used combined quantum mechanics/molecular mechanics (QM/MM) techniques in molecular dynamics simulations and variational transition state theory with multidimensional tunnelling calculations averaged over an ensemble of paths. The results reveal important mechanistic complexity. We calculate activation barriers and KIEs for the two possible proton transfers identified-to either of the carboxylate oxygen atoms of the catalytic base (Asp428beta)-and analyse the contributions of quantum effects. The activation barriers and tunnelling contributions for the two possible proton transfers are similar and lead to a phenomenological activation free energy of 16.5+/-0.9 kcal mol(-1) for transfer to either oxygen (PM3-CHARMM calculations applying PM3-SRP specific reaction parameters), in good agreement with the experimental value of 14.4 kcal mol(-1). In contrast, for the AADH system, transfer to the equivalent OD1 was found to be preferred. The structures of the enzyme complexes during reaction are analysed in detail. The hydrogen bond of Thr474beta(MADH)/Thr172beta(AADH) to the catalytic carboxylate group and the nonconserved active site residue Tyr471beta(MADH)/Phe169beta(AADH) are identified as important factors in determining the preferred oxygen acceptor. The protein environment has a significant effect on the reaction energetics and hence on tunnelling contributions and KIEs. These environmental effects, and the related clearly different preferences for the two carboxylate oxygen atoms (with different KIEs) in MADH/MA and AADH/tryptamine, are possible causes of the differences observed in the KIEs between these two important enzyme reactions.

New Insights into the Reductive Half-reaction Mechanism of Aromatic Amine Dehydrogenase Revealed by Reaction with Carbinolamine Substrates

Aromatic amine dehydrogenase uses a tryptophan tryptophylquinone (TTQ) cofactor to oxidatively deaminate primary aromatic amines. In the reductive half-reaction, a proton is transferred from the substrate C1 to betaAsp-128 O-2, in a reaction that proceeds by H-tunneling. Using solution studies, kinetic crystallography, and computational simulation we show that the mechanism of oxidation of aromatic carbinolamines is similar to amine oxidation, but that carbinolamine oxidation occurs at a substantially reduced rate. This has enabled us to determine for the first time the structure of the intermediate prior to the H-transfer/reduction step. The proton-betaAsp-128 O-2 distance is approximately 3.7A, in contrast to the distance of approximately 2.7A predicted for the intermediate formed with the corresponding primary amine substrate. This difference of approximately 1.0 A is due to an unexpected conformation of the substrate moiety, which is supported by molecular dynamic simulations and reflected in the approximately 10(7)-fold slower TTQ reduction rate with phenylaminoethanol compared with that with primary amines. A water molecule is observed near TTQ C-6 and is likely derived from the collapse of the preceding carbinolamine TTQ-adduct. We suggest this water molecule is involved in consecutive proton transfers following TTQ reduction, and is ultimately repositioned near the TTQ O-7 concomitant with protein rearrangement. For all carbinolamines tested, highly stable amide-TTQ adducts are formed following proton abstraction and TTQ reduction. Slow hydrolysis of the amide occurs after, rather than prior to, TTQ oxidation and leads ultimately to a carboxylic acid product.

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.

Tunneling and Classical Paths for Proton Transfer in an Enzyme Reaction Dominated by Tunneling: Oxidation of Tryptamine by Aromatic Amine Dehydrogenase

Proton tunneling dominates the oxidative deamination of tryptamine catalyzed by the enzyme aromatic amine dehydrogenase. For reaction with the fast substrate tryptamine, a H/D kinetic isotope effect (KIE) of 55 +/- 6 has been reported-one of the largest observed in an enzyme reaction. We present here a computational analysis of this proton-transfer reaction, applying combined quantum mechanics/molecular mechanics (QM/MM) methods (PM3-SRP//PM3/CHARMM22). In particular, we extend our previous computational study (Masgrau et al. Science 2006, 312, 237) by using improved energy corrections, high-level QM/MM methods, and an ensemble of paths to estimate the tunneling contributions. We have carried out QM/MM molecular dynamics simulations and variational transition state theory calculations with small-curvature tunneling corrections. The results provide detailed insight into the processes involved in the reaction. Transfer to the O2 oxygen of the catalytic base, Asp128beta, is found to be the favored reaction both thermodynamically and kinetically, even though O1 is closer in the reactant complex. Comparison of quantum and classical models of proton transfer allows estimation of the contribution of hydrogen tunneling in lowering the barrier to reaction in the enzyme. A reduction of the activation free energy due to tunneling of 3.1 kcal mol-1 is found, which represents a rate enhancement due to tunneling by 2 orders of magnitude. The calculated KIE of 30 is significantly elevated over the semiclassical limit, in agreement with the experimental observations; a semiclassical value of 6 is obtained when tunneling is omitted. A polarization of the C-H bond to be broken is observed due to the close proximity of the catalytic aspartate and the (formally) positively charged imine nitrogen. A comparison is also made with the related quinoprotein methylamine dehydrogenase (MADH)-the much lower KIE of 11 that we obtain for the MADH/methylamine system is found to arise from a more endothermic potential energy surface for the MADH reaction.

Proton Tunneling in Aromatic Amine Dehydrogenase is Driven by a Short-Range Sub-Picosecond Promoting Vibration: Consistency of Simulation and Theory with Experiment

Hydrogen transfer, an essential component of most biological reactions, is a quantum problem. However, the proposed role of compressive motion in promoting enzymatic H-transfer is contentious. Using molecular dynamics simulations and density functional theory (DFT) calculations, we show that, during proton tunneling in the oxidative deamination of tryptamine catalyzed by the enzyme aromatic amine dehydrogenase (AADH), a sub-picosecond promoting vibration is inherent to the iminoquinone intermediate. We show by numerical modeling that this short-range vibration, with a frequency of approximately 165 cm-1, is consistent with "gating" motion in the hydrogen tunneling model of Kuznetsov and Ulstrup (Kuznetsov, A. M.; Ulstrup, J. Can. J. Chem. 1999, 77, 1085) in an enzymatic reaction with an observed protium/deuterium kinetic isotope effect that is not measurably temperature-dependent.

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.

Crystal Structure of an Electron Transfer Complex between Aromatic Amine Dehydrogenase and Azurin fromAlcaligenes 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.