Biosynthesis of topa quinone cofactor in bacterial amine oxidases. Solvent origin of C-2 oxygen determined by Raman spectroscopy.
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.
Nakamura
N
N
Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97291-1000, USA.
Matsuzaki
R
R
Choi
Y H
YH
Tanizawa
K
K
Sanders-Loehr
J
J
D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry / Q000378:metabolism*; D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D001173:Arthrobacter / Q000201:enzymology*; D003001:Cloning, Molecular; D003067:Coenzymes / Q000096:biosynthesis*; D003300:Copper / Q000494:pharmacology; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D004926:Escherichia coli; D007202:Indicators and Reagents; D007553:Isotope Labeling / Q000379:methods; D008744:Methylamines; D010103:Oxygen Isotopes; D010659:Phenylhydrazines; D011994:Recombinant Proteins / Q000378:metabolism; D012997:Solvents; D013059:Spectrum Analysis, Raman; D014867:Water / Q000378:metabolism
D003067:Coenzymes; D007202:Indicators and Reagents; D008744:Methylamines; D010103:Oxygen Isotopes; D010659:Phenylhydrazines; D011994:Recombinant Proteins; D012997:Solvents; D014867:Water; C005307:4-nitrophenylhydrazine; D004295:Dihydroxyphenylalanine; C067626:6-hydroxydopa quinone; D003300:Copper; C027451:methylamine; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase; D006631:Amine Oxidase (Copper-Containing)