New pathway of amine oxidation respiratory chain of Paracoccus denitrificans IFO 12442

Abstract

The physiological electron acceptor of quinohemoprotein amine dehydrogenase (QH-AmDH) from Paracoccus denitrificans IFO 12442 was identified by biochemical and electrochemical methods. Of three types of heme c-containing proteins purified together with QH-AmDH from the periplasm of n-butylamine-grown cells, only constitutive cytochrome c-550 was reduced by the addition of QH-AmDH and n-butylamine. Reconstitution of the respiratory chain revealed that cytochrome c-550 mediates the electron transfer from QH-AmDH to the terminal oxidase. This is a new pathway of the amine oxidation respiratory chain of P. denitrificans. Keywords:bioelectrocatalysis; cytochrome c-550; Paracoccus denitrificans; quinohemoprotein amine dehydrogenase; respiratory chain.Several bacteria are known to utilize primary amines as the sole source of carbon, nitrogen and energy. These substrates are oxidized to the corresponding aldehydes by specific amine dehydrogenases, which function in the periplasmic space of the Gram-negative bacteria. Much effort has been devoted to understanding the structure and function of methylamine dehydrogenase (MADH) [1±5] and aromatic amine dehydrogenase (AADH) [6,7]. Their prosthetic group is known to be tryptophan tryptophylquinone (TTQ). The physiological electron acceptors of these enzymes are proposed to be two different periplasmic type I blue copper proteins: amicyanin for MADH [8,9] and azurin for AADH [10,11]. As extensively studied by Davidson's group, the electron passed to amicyanin from MADH is transferred to the inducible periplasmic cytochrome c-551i [9]. Then the electron from cytochrome c-551i is donated to the membrane-associated respiratory chain (electron-transport system) via the constitutive cytochrome c-550 [12].We have previously demonstrated that Paracoccus denitrificans IFO 12442 produces two types of amine dehydrogenases; one is TTQ-containing MADH mentioned above, and the other is a novel enzyme, quinohemoprotein amine dehydrogenase (QH-AmDH), which contains one heme c besides a quinonoid cofactor in its larger subunit [13]. A similar quinohemoprotein amine dehydrogenase has also been isolated from Pseudomonas putida [14], although the molecular structure of this enzyme is different from that of QH-AmDH from P. denitrificans. For the enzyme from P. putida, the physiologically active electron acceptor has been proposed to be an azurin-like type I blue copper protein, and reconstitution of the amine oxidation system in P. putida has been successfully demonstrated [14].In this study, three kinds of hemoproteins were purified in addition to QH-AmDH from the cell-free extracts of P. denitrificans IFO 12442 grown on n-butylamine. One of the hemoproteins has been found to function as the physiological electron acceptor of QH-AmDH. This paper describes the molecular properties of the hemoprotein to identify it with cytochrome c-550, and the steady-state kinetic parameters of the QH-AmDH-catalyzed reaction with cytochrome c-550 were determined by electrochemical techniques. To study the electr...

Structure of a quinohemoprotein amine dehydrogenase with an uncommon redox cofactor and highly unusual crosslinking

“…Partial reduction of the enzyme by dithionite gave a semiquinone radical EPR signal similar to that of TTQ in MADH. Cytochrome c 550 has been shown to be the natural electron acceptor for QHNDH (11). A similar QHNDH has been isolated from Pseudomonas putida, but apparently with somewhat different subunit composition and with exogenous electron acceptor specificity for azurin (12).…”

The Silent Form of Quinohemoprotein Amine Dehydrogenase fromParacoccus denitrificans

“…16) The purified heme 2 showed two bands, of molecular masses 59.5 kDa and 36.5 kDa, on SDS-PAGE. 16) From these results, heme 2 protein appears to be very similar to QHAmDH in certain physical properties, although it has no catalytic activity for amine oxidation. In this study, we found that heme 2 protein was slowly activated in 10-30 h during incubation with n-butylamine, one of the major substrates of QH-AmDH (Fig.…”

“…QH-AmDH and heme 2 protein from P. denitrificans were isolated and purified as described in the literature. 16) The concentration of the reduced proteins was determined from the absorbance at 552 nm (" ¼ 37:2 mM À1 cm À1 ). The fully oxidized and reduced forms of QH-AmDH and heme 2 were prepared by adding K 3 Fe(CN) 6 and n-butylamine (or Na 2 S 2 O 4 ) respectively.…”

“…One of them, designated heme 2 in the literature, showed two bands, of molecular masses 59.5 kDa and 36.5 kDa, on SDS-PAGE. 16) Surprisingly, this profile is almost identical to that of QH-AmDH, but the heme 2 protein showed no QHAmDH activity. It appeared to be an inactivated form of QH-AmDH.…”

“…16) When cells are grown on methylaminecontaining media, MADH is expressed in P. denitrificans. MADH also catalyzes an interprotein electron transfer of the substrate-derived electrons to cytochrome c-550 via its physiological electron acceptor, amicyanin and cytochrome c-551i.…”