Dept. of Biochemistry and Molecular Biology
Oregon Health & Science University
The copper-containing mine oxidases contain the organic cofactor, 2,4,5- trihydroxyphenylalanine (TPQ), whose C5=0 participates in the oxidative deamination of primary amines. Differing rates of solvent exchange for the C5=O and C3-H of the TPQ cofactor have been observed by resonance Raman (RR) spectroscopy in different amine oxidases. Rapid C5=0 exchange and slow or nonobservable C3-H exchange lead to the prediction of a productive TPQ orientation for the mine oxidases from Hansenula polymorpha (HPAO), Arthrobacter globifomis (AGAO), pea seedling (PSAO) at pH 7.1, and the E406Q-HPAO mutant. In contrast, slow C5=O exchange and fast C3-H exchange leads to the prediction of a flipped TPQ orientation for PSAO at low pH, and the D319E-HPAO and D319N-HPAO mutants. Finally, for the amine oxidase from Escherichia coli (ECAO) and the N404A-HPAO mutant, a mobile cofactor is predicted. RR spectroscopy has also been used to identify the dead-end complexes formed during catalytic turnover with HPAO mutants. Thus, the flanking-residue mutant, N404A, reacts with methylamine to form a deprotonated product Schiff base. The catalytic-base mutants, D319E and D319N, form a deprotonated iminoquinone species with methylamine and ammonium chloride, respectively. The deprotonated state of these intermediates is indicative of a flipped cofactor orientation (with C5 directed away from the substrate-binding pocket), thereby explaining their dead-end character. RR spectroscopy of the heme cofactor in cystathionine Ã-synthase has enabled us to identify the heme axial ligands as His and Cys in both the ferrous and ferric states. The Cys ligand was elucidated through global [superscript 34]S substitution and the identification of the Fe-S(Cys) stretch at 3 12 cm[superscript -1] (-3 cm[superscript -1] in [superscript 34]S). The His ligand was verified through examination of the ferrous-CO complex where the v(Fe-CO) and v(C0) frequencies at 497 and 1961 cm[superscript -1], respectively, correlate with a neutral His acting as the sixth ligand. Additionally, we found that the substrate homocysteine does not covalently bind to the heme, suggesting that the heme may be playing a regulatory role.
OGI School of Science and Engineering
Green, Edward L., "Characterization of the TPQ cofactor in amine oxidases and the heme cofactor in cystathionine beta-synthase by resonance raman spectroscopy. Implications for catalytic properties" (2001). Scholar Archive. 119.