4-nitrophenylhydrazine and 6-hydroxydopa-quinone

Resonance Raman spectroscopy is an excellent technique for providing structural information on the 2,4, 5-trihydroxyphenylalanine quinone (TPQ) cofactor in copper-containing amine oxidases. This technique has been used to investigate the copper- and O2-dependent biosynthesis of the TPQ cofactor in phenylethylamine oxidase (PEAO) and histamine oxidase from Arthrobacter globiformis. Incubation of the holoenzyme in H218O causes frequency shifts at 1684(-26) cm-1 in PEAO and at 1679(-28) cm-1 in histamine oxidase, allowing this feature to be assigned to the C=O stretch of a single carbonyl group at the C-5 position. When apoprotein is reacted with Cu(II) and O2 in the presence of H218O, the resultant holoproteins show increased shifts of -3 to -6 cm-1 in a number of other vibrational modes, particularly at 411 and 1397 cm-1. Because these small shifts persist when the H218O-regenerated protein is back-exchanged into H216O, they can be assigned to oxygen isotope substitution at the C-2 postion. No isotope shifts are observed when apoprotein is regenerated with Cu(II) in the presence of 18O2. Thus, it is concluded that the C-2 oxygen atom of TPQ originates from H2O rather than O2. The isotope dependence of the 1397-cm-1 mode allows it to be assigned to the C O moiety at the C-2 position, with its low frequency being indicative of only partial double bond character. Similar frequency shifts due to 18O at C-2 are observed in the resonance Raman spectra of H218O-regenerated PEAO after derivatization of the C-5 carbonyl with either p-nitrophenylhydrazine (-5 cm-1 at 480 cm-1) or methylamine (-5 cm-1 at 1301 cm-1). Taken together, these results indicate that the TPQ cofactor in the native enzyme has substantial electron delocalization between the C-2 and C-4 oxygens and that only the C-5 oxygen has predominantly C=O character.

Topics: Amine Oxidase (Copper-Containing); Amino Acid Oxidoreductases; Arthrobacter; Cloning, Molecular; Coenzymes; Copper; Dihydroxyphenylalanine; Escherichia coli; Indicators and Reagents; Isotope Labeling; Methylamines; Oxygen Isotopes; Phenylhydrazines; Recombinant Proteins; Solvents; Spectrum Analysis, Raman; Water

Amine oxidases (EC 1.4.3.6) from Aspergillus niger, AO-I (2 x 75 kDa) and AO-II (80 kDa), were examined to determine the cofactor structure. Inactivated with p-nitrophenylhydrazine, they showed absorption and fluorescence spectra similar to those published for other copper amine oxidases and to topa hydantoin p-nitrophenylhydrazone. After digestion by thermolysin and pronase, cofactor peptides were purified by HPLC and sequenced. For thermolytic peptides, a typical topa consensus sequence, Asn-X-Glu-Tyr, was obtained for AO-II, although in case of AO-I it overlapped with Val-Val-Ile-Glu-Pro-Tyr-Gly. For pronase peptides of AO-I, only the latter sequence was obtained. NMR and mass spectroscopy confirmed the residue X as topa p-nitrophenylhydrazone in AO-II and revealed the presence of a residue Z attached to the Glu in the peptide Val-Val-Ile-Glu(Z)-Pro of AO-I. This residue was separated from the peptide by hydrolysis and identified as a product derived from topa quinone. The data, together with amino-acid sequence of AO-I, confer strong evidence for topa quinone as the cofactor, bound in the typical consensus sequence. Raman spectra of the p-nitrophenylhydrazone derivative of AO-I and its pronase peptide showed essentially the same peaks matching to a model compound for topa p-nitrophenylhydrazone. However, there may exist an unusual ester link between the topa-404 and Glu-145 in the native enzyme.

Topics: Amine Oxidase (Copper-Containing); Amino Acid Sequence; Aspergillus niger; Coenzymes; Consensus Sequence; Dihydroxyphenylalanine; Glutamic Acid; Models, Chemical; Molecular Sequence Data; Oxidoreductases Acting on CH-NH Group Donors; Peptide Fragments; Phenylhydrazines; Sequence Analysis

Pig kidney diamine oxidase was purified to homogeneity. The reaction product of the cofactor with p-nitrophenylhydrazine (pNPH) was liberated with pronase treatment and purified. 1H NMR, uv/vis, and electrospray tandem mass spectroscopy revealed it to be a dipeptide with the sequence topaquinone-pNPH and aspartate. No heterogeneity was observed, indicating that no intramolecular cyclization of the quinone moiety occurs in the time span of the isolation and of the measurements. Similar results were obtained with the more widely applicable reagent, phenylhydrazine, and using the aromatic amine oxidase from Escherichia coli. From the amount and ease with which the dipeptide could be isolated, the procedure used here is more convenient than the existing one for the identification of protein-integrated quinone cofactors.

Topics: Amine Oxidase (Copper-Containing); Amino Acid Sequence; Animals; Consensus Sequence; Dihydroxyphenylalanine; Escherichia coli; Indicators and Reagents; Kidney; Magnetic Resonance Spectroscopy; Mass Spectrometry; Molecular Sequence Data; Molecular Structure; Oxidoreductases Acting on CH-NH Group Donors; Phenylhydrazines; Spectrophotometry; Swine