The silent form of quinohemoprotein amine dehydrogenase from paracoccus denitrificans

Abstract

Quinohemoprotein amine dehydrogenase (QHAmDH) from Paracoccus denitrificans is an αβγ heterotrimeric enzyme catalyzing oxidative deamination of amines with large substitutes, such as butylamine and benzylamine. The smallest γ subunit has cross-linking cysteine tryptophylquinone (CTQ) as a catalytic center. A hemoprotein similar to QH-AmDH in molecular mass, subunit structure, and UV-vis spectral property, but having no enzymatic activity, was isolated. The enzymatically silent form (sQH-AmDH) was activated slowly by the substrates of QH-AmDH, and quickly by reductants, dithionite and dithiothreitol. Electrolysis of sQH-AmDH yielded the active form at potentials more negative than -0:17V (vs. SHE). The activated protein reacted with a carbonyl reagent, 4-nitrophenylhydrazine, giving a typical spectrum of 4-nitrophenylhydrazone, while sQH-AmDH did not give such a spectrum. The γ subunit of sQH-AmDH showed a sharp peak at 390nm in UV-vis spectrum clearly distinguishable from that of QH-AmDH. Electrospray ionization mass spectrometric analysis showed that the molecular mass of the γ subunit of sQH-AmDH was larger than that of QH-AmDH by 15.6. The data suggest that the CTQ-like moiety of sQH-AmDH is an oxime. This hypothesis was confirmed by subsequent hydroxylamine treatment of QH-AmDH. QH-AmDH was treated with hydroxylamine yielding an oxime derivative. The UV-vis spectral properties of the γ subunit of hydroxylamine-treated QH-AmDH were identical to those of sQH-AmDH. The hydroxylamine-treated QH-AmDH was also reactivated by butylamine, as in the case of sQH-AmDH.