The crystal structures of the copper enzyme phenylethylamine oxidase from the Gram-positive bacterium Arthrobacter globiformis (AGAO) have been determined and refined for three forms of the enzyme: the holoenzyme in its active form (at 2.2 A resolution), the holoenzyme in an inactive form (at 2.8 A resolution), and the apoenzyme (at 2.2 A resolution). The holoenzyme has a topaquinone (TPQ) cofactor formed from the apoenzyme by the post-translational modification of a tyrosine residue in the presence of Cu2+. Significant differences between the three forms of AGAO are limited to the active site. The polypeptide fold is closely similar to those of the amine oxidases from Escherichia coli [Parsons, M. R., et al. (1995) Structure 3, 1171-1184] and pea seedlings [Kumar, V., et al. (1996) Structure 4, 943-955]. In the active form of holo-AGAO, the active-site Cu atom is coordinated by three His residues and two water molecules in an approximately square-pyramidal arrangement. In the inactive form, the Cu atom is coordinated by the same three His residues and by the phenolic oxygen of the TPQ, the geometry being quasi-trigonal-pyramidal. There is evidence of disorder in the crystals of both forms of holo-AGAO. As a result, only the position of the aromatic group of the TPQ cofactor, but not its orientation about the Cbeta-Cgamma bond, is determined unequivocally. In apo-AGAO, electron density consistent with an unmodified Tyr occurs at a position close to that of the TPQ in the inactive holo-AGAO. This observation has implications for the biogenesis of TPQ. Two features which have not been described previously in amine oxidase structures are a channel from the molecular surface to the active site and a solvent-filled cavity at the major interface between the two subunits of the dimer.

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In studies of the chemistry of new copper−nitrosyl complexes supported by tris(3-(trifluoromethyl)-5-methylpyrazol-1-yl)hydroborate (TpCF 3 ,CH 3 ) and tris(3-mesitylpyrazol-1-yl)hydroborate (TpMs,H), significant effects of the scorpionate ligand substituents on the properties of the {CuNO}11 unit were found that have implications for environmental influences on similar species in biological and catalytic systems. The copper(I) complexes TpMs,HCu(THF) and TpCF 3 ,CH 3 Cu(CH3CN) were structurally characterized by X-ray crystallography, and their respective CO and NO adducts were studied by FTIR, EPR, NMR, and/or UV−vis spectroscopies in solution. Both nitrosyl complexes disproportionate in the presence of excess NO to N2O and TpR,R‘Cu(NO2); an X-ray structure of the latter product supported by TpCF 3 ,CH 3 was determined. Unlike previously studied paramagnetic [CuNO]11 compounds that exhibit EPR signals with g < 2.0 and large A NO values at temperatures below ∼40 K (Ruggiero, C. E.; Carrier, S. M.; Antholine, W. E.; Whittaker, J. W.; Cramer, C. J.; Tolman, W. B. J. Am. Chem. Soc. 1993, 115, 11285−11298), TpMs,HCu(NO) is EPR silent at 4.2 K and exhibits an NMR spectrum (238 K, toluene-d 8) with sharp signals. Peak assignments for the NMR spectrum were deduced from integrated intensities, temperature-dependent isotropic shifts, and the nuclear relaxation rates. The unique NMR spectral behavior for the TpMs,H complex, which only differs from those of analogues with simple phenyl substituents by virtue of the shape of the substrate binding pocket enforced by the mesityl methyl groups, suggests that caution should be exercised in characterizing such adducts in proteins and heterogeneous systems; subtle environmental effects may determine the applicability of EPR versus NMR methods. The electron-withdrawing effects of the trifluoromethyl substituents in TpCF 3 ,CH 3 Cu(NO) perturb ν(NO) and the Cu(I) → NO MLCT energy in the respective FTIR and UV−vis spectra and induce a significant slowing of its disproportionation rate. These results, in conjunction with those obtained from kinetic and spectroscopic studies on the TpMs,H system, support a mechanism for the disproportionation involving generation of the CuNO adduct from NO and the Cu(I) precursor in a preequilibrium step, followed by electrophilic attack of a second NO molecule on the adduct that is rate-controlling.

The siderophores of Bacillus anthracis are critical for the pathogen's proliferation and may be necessary for its virulence. Bacillus anthracis str. Sterne cells were cultured in iron free media and the siderophores produced were isolated and purified using a combination of XAD-2 resin, reverse-phase FPLC, and size exclusion chromatography. A combination of 1H and 13C NMR spectroscopy, UV spectroscopy and ESI-MS/MS fragmentation were used to identify the primary siderophore as petrobactin, a catecholate species containing unusual 3,4-dihydroxybenzoate moieties, previously only identified in extracts of Marinobacter hydrocarbonoclasticus. A secondary siderophore was observed and structural analysis of this species is consistent with that reported for bacillibactin, a siderophore observed in many species of bacilli. This is the first structural characterization of a siderophore from B. anthracis, as well as the first characterization of a 3,4-DHB containing catecholate in a pathogen.

Nitrito‐O,O‐copper complexes and dinitrogen oxide are the products of the reaction of excess NO with the nitrosyl complexes [LCu(NO)] (L = substituted tris(pyrazolyl)hydroborate), a process that models environmentally important transformations carried out by copper ions in proteins and zeolites. According to the results of kinetic studies, the involvement of dimeric copper species in the rate‐determining step is ruled out, which suggests that NO coupling occurs at a single metal ion.

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An alternative purification for apophenylethylamine oxidase from Arthrobacter globiformis has been developed, which avoids the use of possible contaminants that may interfere with the topa quinone (TPQ) self-processing reaction. The binding of Cu(II) and the kinetics of TPQ formation in these enzyme preparations have been reinvestigated. Our results show that Cu(II) is not significantly reduced when added to the apoprotein under anaerobic conditions. The Cu(II) EPR and circular dichroism spectra of the initially formed complex are different from the spectra of the mature Cu(II)/TPQ-containing protein, indicating that the active site structure must be altered during TPQ formation. The kinetics we observe are cleanly first-order in protein [measured subsequent to Cu(II) binding] when dioxygen is present in pseudo-first-order excess (k(obs) = 1.5 min(-1)). We found no rate dependence on copper, so long as one copper per subunit was present. This indicates that tyrosine oxidation to give TPQ depends only on the copper that is bound in the active site. These results differ from those originally reported; an alternative mechanism, which involves attack of an activated copper-oxygen species on a tyrosine radical intermediate, is proposed for TPQ formation.

Uptake of plutonium and uranium mediated by the siderophore desferrioxamine-B (DFOB) has been studied for the common soil aerobe Microbacterium flavescens(JG-9). M. flavescens does not bind or take up nitrilotriacetic acid (NTA) complexes of U(VI), Fe(III), or Pu(IV) or U(VI)-DFOB but does take up Fe(III)-DFOB and Pu(IV)-DFOB. Pu(IV)-DFOB and Fe(III)-DFOB accumulations are similar: only living and metabolically active bacteria take up these metal-siderophore complexes. The Fe(III)-DFOB and Pu(IV)-DFOB complexes mutually inhibit uptake of the other, indicating that they compete for shared binding sites or uptake proteins. However, Pu uptake is much slower than Fe uptake, and cumulative Pu uptake is less than Fe, 1.0 nmol of Fe vs 0.25 nmol of Pu per mg of dry weight bacteria. The Pu(IV)-DFOB interactions with M. flavescens suggest that Pu-siderophore complexes could generally be recognized by Fe-siderophore uptake systems of many bacteria, fungi, or plants, thereby affecting Pu environmental mobility and distribution. The results also suggest that the siderophore complexes of tetravalent metals can be recognized by Fe-siderophore uptake proteins.