Characterization of the Preprocessed Copper Site Equilibrium in Amine Oxidase and Assignment of the Reactive Copper Site in Topaquinone Biogenesis.

Copper-dependent amine oxidases produce their redox active cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), via the CuII-catalyzed oxygenation of an active site tyrosine. This study addresses possible mechanisms for this biogenesis process by presenting the geometric and electronic structure characterization of the CuII-bound, prebiogenesis (preprocessed) active site of the enzyme Arthrobacter globiformis amine oxidase (AGAO). CuII-loading into the preprocessed AGAO active site is slow ( kobs = 0.13 h-1), and is preceded by CuII binding in a separate kinetically favored site that is distinct from the active site. Preprocessed active site CuII is in a thermal equilibrium between two species, an entropically favored form with tyrosine protonated and unbound from the CuII site, and an enthalpically favored form with tyrosine bound deprotonated to the CuII active site. It is shown that the CuII-tyrosinate bound form is directly active in biogenesis. The electronic structure determined for the reactive form of the preprocessed CuII active site is inconsistent with a biogenesis pathway that proceeds through a CuI-tyrosyl radical intermediate, but consistent with a pathway that overcomes the spin forbidden reaction of 3O2 with the bound singlet substrate via a three-electron concerted charge-transfer mechanism.

Cobalt substitution supports an inner-sphere electron transfer mechanism for oxygen reduction in pea seedling amine oxidase.

Copper amine oxidases (CAOs) are a large family of proteins that use molecular oxygen to oxidize amines to aldehydes with the concomitant production of hydrogen peroxide and ammonia. CAOs utilize two cofactors for this reaction: topaquinone (TPQ) and a Cu(II) ion. Two mechanisms for oxygen reduction have been proposed for these enzymes. In one mechanism (involving inner-sphere electron transfer to O(2)), Cu(II) is reduced by TPQ, forming Cu(I), to which O(2) binds, forming a copper-superoxide complex. In an alternative mechanism (involving outer-sphere electron transfer to O(2)), O(2) is directly reduced by TPQ, without reduction of Cu(II). Substitution of Cu(II) with Co(II) has been used to distinguish between the two mechanisms in several CAOs. Because it is unlikely that Co(II) could be reduced to Co(I) in this environment, an inner-sphere mechanism, as described above, is prevented. We adapted metal replacement methods used for other CAOs to the amine oxidase from pea seedlings (PSAO). Cobalt-substituted PSAO (CoPSAO) displayed nominal catalytic activity: k(cat) is 4.7% of the native k(cat), and K(M) (O(2)) for CoPSAO is substantially (22-fold) higher. The greatly reduced turnover number for CoPSAO suggests that PSAO uses the inner-sphere mechanism, as has been predicted from (18)O isotope effect studies (Mukherjee et al. in J Am Chem Soc 130:9459-9473, 2008), and is catalytically compromised when constrained to operate via outer-sphere electron transfer to O(2). This study, together with previous work, provides strong evidence that CAOs use both proposed mechanisms, but each homolog may prefer one mechanism over the other.

Spectroscopic identification of heme axial ligands in HtsA that are involved in heme acquisition by Streptococcus pyogenes.

The heme-binding proteins Shp and HtsA of Streptococcus pyogenes are part of the heme acquisition machinery in which Shp directly transfers its heme to HtsA. Mutagenesis and spectroscopic analyses were performed to identify the heme axial ligands in HtsA and to characterize axial mutants of HtsA. Replacements of the M79 and H229 residues, not the other methionine and histidine residues, with alanine convert UV-vis spectra of HtsA with a low-spin, hexacoordinate heme iron into spectra of high-spin heme complexes. Ferrous M79A and H229A HtsA mutants possess magnetic circular dichroism (MCD) spectra that are similar with those of proteins with pentacoordinate heme iron. Ferric M79A HtsA displays UV-vis, MCD, and resonance Raman (RR) spectra that are typical of a hexacoordinate heme iron with histidine and water ligands. In contrast, ferric H229A HtsA has UV-vis, MCD, and RR spectra that represent a pentacoordinate heme iron complex with a methionine axial ligand. Imidazole readily forms a low-spin hexacoordinate adduct with M79A HtsA with a K(d) of 40.9 muM but not with H229A HtsA, and cyanide binds to M79A and H229A with K(d) of 0.5 and 19.1 microM, respectively. The ferrous mutants rapidly bind CO and form simple CO complexes. These results establish the H229 and M79 residues as the axial ligands of the HtsA heme iron, indicate that the M79 side is more accessible to the solvent than the H229 side of the bound heme in HtsA, and provide unique spectral features for a protein with pentacoordinate, methionine-ligated heme iron. These findings will facilitate elucidation of the molecular mechanism and structural basis for rapid and direct heme transfer from Shp to HtsA.

Structure and inhibition of human diamine oxidase.

Humans have three functioning genes that encode copper-containing amine oxidases. The product of the AOC1 gene is a so-called diamine oxidase (hDAO), named for its substrate preference for diamines, particularly histamine. hDAO has been cloned and expressed in insect cells and the structure of the native enzyme determined by X-ray crystallography to a resolution of 1.8 A. The homodimeric structure has the archetypal amine oxidase fold. Two active sites, one in each subunit, are characterized by the presence of a copper ion and a topaquinone residue formed by the post-translational modification of a tyrosine. Although hDAO shares 37.9% sequence identity with another human copper amine oxidase, semicarbazide sensitive amine oxidase or vascular adhesion protein-1, its substrate binding pocket and entry channel are distinctly different in accord with the different substrate specificities. The structures of two inhibitor complexes of hDAO, berenil and pentamidine, have been refined to resolutions of 2.1 and 2.2 A, respectively. They bind noncovalently in the active-site channel. The inhibitor binding suggests that an aspartic acid residue, conserved in all diamine oxidases but absent from other amine oxidases, is responsible for the diamine specificity by interacting with the second amino group of preferred diamine substrates.

Inner-sphere mechanism for molecular oxygen reduction catalyzed by copper amine oxidases.

Copper and topaquinone (TPQ) containing amine oxidases utilize O2 for the metabolism of biogenic amines while concomitantly generating H2O2 for use by the cell. The mechanism of O2 reduction has been the subject of long-standing debate due to the obscuring influence of a proton-coupled electron transfer between the tyrosine-derived TPQ and copper, a rapidly established equilibrium precluding assignment of the enzyme in its reactive form. Here, we show that substrate-reduced pea seedling amine oxidase (PSAO) exists predominantly in the Cu(I), TPQ semiquinone state. A new mechanistic proposal for O2 reduction is advanced on the basis of thermodynamic considerations together with kinetic studies (at varying pH, temperature, and viscosity), the identification of steady-state intermediates, and the analysis of competitive oxygen kinetic isotope effects, (18)O KIEs, [kcat/KM((16,16)O2)]/[kcat/KM((16,18)O2)]. The (18)O KIE = 1.0136 +/- 0.0013 at pH 7.2 is independent of temperature from 5 degrees C to 47 degrees C and insignificantly changed to 1.0122 +/- 0.0020 upon raising the pH to 9, thus indicating the absence of kinetic complexity. Using density functional methods, the effect is found to be precisely in the range expected for reversible O2 binding to Cu(I) to afford a superoxide, [Cu(II)(eta(1)-O2)(-I)](+), intermediate. Electron transfer from the TPQ semiquinone follows in the first irreversible step to form a peroxide, Cu(II)(eta(1)-O2)(-II), intermediate driving the reduction of O2. The similar (18)O KIEs reported for copper amine oxidases from other sources raise the possibility that all enzymes react by related inner-sphere mechanisms although additional experiments are needed to test this proposal.

Enantiomer-specific binding of ruthenium(II) molecular wires by the amine oxidase of Arthrobacter globiformis.

The copper amine oxidase from Arthrobacter globiformis (AGAO) is reversibly inhibited by molecular wires comprising a Ru(II) complex head group and an aromatic tail group joined by an alkane linker. The crystal structures of a series of Ru(II)-wire-AGAO complexes differing with respect to the length of the alkane linker have been determined. All wires lie in the AGAO active-site channel, with their aromatic tail group in contact with the trihydroxyphenylalanine quinone (TPQ) cofactor of the enzyme. The TPQ cofactor is consistently in its active ("off-Cu") conformation, and the side chain of the so-called "gate" residue Tyr296 is consistently in the "gate-open" conformation. Among the wires tested, the most stable complex is produced when the wire has a -(CH2)4- linker. In this complex, the Ru(II)(phen)(bpy)2 head group is level with the protein molecular surface. Crystal structures of AGAO in complex with optically pure forms of the C4 wire show that the linker and head group in the two enantiomers occupy slightly different positions in the active-site channel. Both the Lambda and Delta isomers are effective competitive inhibitors of amine oxidation. Remarkably, inhibition by the C4 wire shows a high degree of selectivity for AGAO in comparison with other copper-containing amine oxidases.

Direct electron transfer from pseudoazurin to nitrous oxide reductase in catalytic N2O reduction.

Pseudoazurin (PAz), a well-characterized blue copper electron-transfer protein, is shown herein to be capable of mediating electron transfer to the nitrous oxide reductase (N(2)OR) from Achromobacter cycloclastes (Ac). Spectroscopic measurements demonstrate that reduced PAz is efficiently re-oxidized by a catalytic amount of N(2)OR in the presence of N(2)O. Fits of the kinetics resulted in K(M) (N(2)O) and k(cat) values of 19.1±3.8 µM and 89.3±4.2s(-1) respectively. The K(M) (PAz) was 28.8±6.6 µM. The electrochemistry of Ac pseudoazurin (AcPAz) in the presence of Ac nitrous oxide reductase (AcN(2)OR) and N(2)O displayed an enhanced cathodic sigmoidal current-potential curve, in excellent agreement with the re-oxidation of reduced AcPAz during the catalytic reduction of N(2)O by AcN(2)OR. Modeling the structure of the AcPAz-AcN(2)OR electron transfer complex indicates that AcPAz binds near Cu(A) in AcN(2)OR, with parameters consistent with the formation of a transient, weakly-bound complex. Multiple, potentially efficient electron-transfer pathways between the blue-copper center in AcPAz and Cu(A) were also identified. Collectively, the data establish that PAz is capable of donating electrons to N(2)OR in N(2)O reduction and is a strong candidate for the physiological electron donor to N(2)OR in Ac.

Non-covalent interactions in blue copper protein probed by Met16 mutation and electronic and resonance Raman spectroscopy of Achromobacter cycloclastes pseudoazurin.

We have used low-temperature (77K) resonance Raman (RR) spectroscopy as a probe of the electronic and molecular structure to investigate weak pi-pi interactions between the metal ion-coordinated His imidazoles and aromatic side chains in the second coordination sphere of blue copper proteins. For this purpose, the RR spectra of Met16 mutants of Achromobacter cycloclastes pseudoazurin (AcPAz) with aromatic (Met16Tyr, Met16Trp, and Met16Phe) and aliphatic (Met16Ala, Met16Val, Met16Leu, and Met16Ile) amino acid side chains have been obtained and analyzed over the 100-500cm(-1) spectral region. Subtle strengthening of the Cu(II)-S(Cys) interaction on replacing Met16 with Tyr, Trp, and Phe is indicated by the upshifted (0.3-0.8cm(-1)) RR bands involving nu(Cu-S)(Cys) stretching modes. In contrast, the RR spectra of Met16 mutants with aliphatic amino acids revealed larger (0.2-1.8cm(-1)) shifts of the nu(Cu-S)(Cys) stretching modes to a lower frequency region, which indicate a weakening of the Cu(II)-S(Cys) bond. Comparisons of the predominantly nu(Cu-S)(Cys) stretching RR peaks of the Met16X=Tyr, Trp, and Phe variants, with the molar absorptivity ratio epsilon(1)/epsilon(2) of sigma( approximately 455nm)/pi( approximately 595nm) (Cys)S-->Cu(II) charge-transfer bands in the optical spectrum and the axial/rhombic EPR signals, revealed a slightly more trigonal disposition of ligands about the copper(II) ion. In contrast, the RR spectra of Met16Z=Ala, Val, Leu, and Ile variants with aliphatic amino acid side chains show a more tetrahedral perturbation of the copper active site, as judged by the lower frequencies of the nu(Cu-S)(Cys) stretching modes, much larger values of the epsilon(1)/epsilon(2) ratio, and the increased rhombicity of the EPR spectra.

Pi-pi interaction between aromatic ring and copper-coordinated His81 imidazole regulates the blue copper active-site structure.

Noncovalent weak interactions play important roles in biological systems. In particular, such interactions in the second coordination shell of metal ions in proteins may modulate the structure and reactivity of the metal ion site in functionally significant ways. Recently, pi-pi interactions between metal ion coordinated histidine imidazoles and aromatic amino acids have been recognized as potentially important contributors to the properties of metal ion sites. In this paper we demonstrate that in pseudoazurin (a blue copper protein) the pi-pi interaction between a coordinated histidine imidazole ring and the side chains of aromatic amino acids in the second coordination sphere, significantly influences the properties of the blue copper site. Electronic absorption and electron paramagnetic resonance spectra indicate that the blue copper electronic structure is perturbed, as is the redox potential, by the introduction of a second coordination shell pi-pi interaction. We suggest that the pi-pi interaction with the metal ion coordinated histidine imidazole ring modulates the electron delocalization in the active site, and that such interactions may be functionally important in refining the reactivity of blue copper sites.

The Formation of lysine tyrosylquinone (LTQ) is a self-processing reaction. Expression and characterization of a Drosophila lysyl oxidase.

Recent work in our laboratory has established methods for the expression and purification of a recombinant form of Drosophila lysyl oxdidase (rDMLOXL-1) [Molnar, J., Ujfaludi, Z., Fong, S. F. T., Bollinger, J. A., Waro, G., Fogelgren, B., Dooley, D. M., Mink, M., and Csiszar, K. (2005) J. Biol. Chem. 280, 22977-22985]. Previous investigations on the expression and purification of recombinant forms of lysyl oxidase [Kagan, H. M., Reddy, V. B., Panchenko, M. V., Nagan, N., Boak, A. M., Gacheru, S. N., and Thomas, K. (1995) J. Cell. Biochem. 59, 329-338] and lysyl oxidase-like proteins [Jung, S. T., Kim, M. S., Seo, J. Y., Kim, H. C., and Kim, Y. (2003) Protein Expression Purif. 31, 240-246] [Molnar, J., Fong, K. S. K., He, Q. P., Hayashi, K., Kim, Y., Fong, S. F. T., Fogelgren, B., Szauter, K. M., Mink, M., and Csiszar, K. (2003) Biochim. Biophys. Acta 1647, 220-224] have been reported in the literature. However, this is the first time that an expression system has been developed yielding sufficient amounts of a recombinant lysyl oxidase for detailed characterization. rDmLOXL-1 is secreted into the medium from S2 cells, and the protein is readily purified by Cibacon blue affinity chromatography yielding 10 mg of protein per liter of medium. The protein, as initially purified, is inactive and has no detectable copper or cofactor present. Following aerobic dialysis against copper, the protein is active and displays an electronic absorption spectrum with lambda(max) at 504 nm, consistent with the presence of an organic cofactor. Addition of phenylhydrazine to the copper-loaded protein produced a high-affinity adduct with lambda(max) at 454 nm. Comparison of the resonance Raman spectra of this adduct and a phenylhydrazine-labeled model compound of lysine tyrosylquinone (LTQ) establishes that the cofactor in the active, copper-containing enzyme is LTQ. Collectively, the data demonstrate that LTQ biogenesis most likely occurs by self-processing chemistry, requiring only the precursor protein, copper, and oxygen. Electron paramagnetic resonance and circular dichroism spectroscopy were used to characterize the Cu(II) site in rDmLOXL-1. The data are consistent with a tetragonal Cu(II) site with nitrogen and oxygen ligands. Recombinant DmLOXL-1 displayed significant activity toward tropoelastin and a wide variety of amines including polyamines and diamines. beta-aminoproprionitrile (betaAPN), a well-known irreversible inhibitor of mammalian lysyl oxidases, is also a potent inhibitor of rDmLOXL-1. Results from this investigation have important implications for the lysyl oxidase family.

Role of the interactions between the active site base and the substrate Schiff base in amine oxidase catalysis. Evidence from structural and spectroscopic studies of the 2-hydrazinopyridine adduct of Escherichia coli amine oxidase.

2-Hydrazinopyridine (2HP) is an irreversible inhibitor of copper amine oxidases (CAOs). 2HP reacts directly at the C5 position of the TPQ cofactor, yielding an intense chromophore with lambda(max) approximately 430 nm (adduct I) in Escherichia coli amine oxidase (ECAO). The adduct I form of wild type (WT-ECAO) was assigned as a hydrazone on the basis of the X-ray crystal structure. The hydrazone adduct appears to be stabilized by two key hydrogen-bonding interactions between the TPQ-2HP moiety and two active site residues: the catalytic base (D383) and the conserved tyrosine residue (Y369). In this work, we have synthesized a model compound (2) for adduct I from the reaction of a TPQ model compound (1) and 2HP. NMR spectroscopy and X-ray crystallography show that 2 exists predominantly as the azo form (lambda(max) at 414 nm). Comparison of the UV-vis and resonance Raman spectra of 2 with adduct I in WT, D383E, D383N, and Y369F forms of ECAO revealed that adduct I in WT and D383N is a tautomeric mixture where the hydrazone form is favored. In D383E adduct I, the equilibrium is further shifted in favor of the hydrazone form. UV-vis spectroscopic pH titrations of adduct I in WT, D383N, D383E, and 2 confirmed that D383 in WT adduct I is protonated at pH 7 and stabilizes the hydrazone tautomer by a short hydrogen-bonding interaction. The deprotonation of D383 (pKa approximately 9.7) in adduct I resulted in conversion of adduct I to the azo tautomer with a blue shift of the lambda(max) to 420 nm, close to that of 2. In contrast, adduct I in D383N and D383E is stable and did not show any pH-dependent spectral changes. In Y369F, adduct I was not stable and gradually converted into a new species with lambda(max) at approximately 530 nm (adduct II). A detailed mechanism for the adduct I formation in WT has been proposed that is consistent with the mechanism proposed for the oxidation of substrate by CAOs but addresses some key differences in the active site chemistry of hydrazine inhibitors and substrate amines.

Active site rearrangement of the 2-hydrazinopyridine adduct in Escherichia coli amine oxidase to an azo copper(II) chelate form: a key role for tyrosine 369 in controlling the mobility of the TPQ-2HP adduct.

Adduct I (lambda(max) at approximately 430 nm) formed in the reaction of 2-hydrazinopyridine (2HP) and the TPQ cofactor of wild-type Escherichia coli copper amine oxidase (WT-ECAO) is stable at neutral pH, 25 degrees C, but slowly converts to another spectroscopically distinct species with a lambda(max) at approximately 530 nm (adduct II) at pH 9.1. The conversion was accelerated either by incubation of the reaction mixture at 60 degrees C or by increasing the pH (>13). The active site base mutant forms of ECAO (D383N and D383E) showed spectral changes similar to WT when incubated at 60 degrees C. By contrast, in the Y369F mutant adduct I was not stable at pH 7, 25 degrees C, and gradually converted to adduct II, and this rate of conversion was faster at pH 9. To identify the nature of adduct II, we have studied the effects of pH and divalent cations on the UV-vis and resonance Raman spectroscopic properties of the model compound of adduct I (2). Strikingly, it was found that addition of Cu2+ to 2 at pH 7 gave a product (3) that exhibited almost identical spectroscopic signatures to adduct II. The X-ray crystal structure of 3 shows that it is the copper-coordinated form of 2, where the +2 charge of copper is neutralized by a double deprotonation of 2. These results led to the proposal that adduct II in the enzyme is TPQ-2HP that has migrated onto the active site Cu2+. The X-ray crystal structure of Y369F adduct II confirmed this assignment. Resonance Raman and EPR spectroscopy showed that adduct II in WT-ECAO is identical to that seen in Y369F. This study clearly demonstrates that the hydrogen-bonding interaction between O4 of TPQ and the conserved Tyr (Y369) is important in controlling the position and orientation of TPQ in the catalytic cycle, including optimal orientation for reactivity with substrate amines.