The Conversion from the Dehydrogenase Type to the Oxidase Type of Rat Liver Xanthine Dehydrogenase by Modification of Cysteine Residues with Fluorodinitrobenzene

Abstract

When rat liver xanthine dehydrogenase was incubated with fluorodinitrobenzene (FDNB) at pH 8.5, the total enzyme activity decreased gradually to a limited value of initial activity with modification of two lysine residues in a similar way to the modification of bovine milk xanthine oxidase with FDNB (Nishino, T., Tsushima, K., Hille, R. and Massey, V. (1982) J. Biol. Chem. 257, 7348 -7353). After modification with FDNB, the two peptides containing dinitrophenyl-lysine were isolated from the molybdopterin domain after proteolytic digestion and were identified as Lys 754 and Lys 771by sequencing the peptides. During the modification of these lysine residues, xanthine dehydrogenase was found to be converted to an oxidase form in the early stage of incubation. Incorporation of the 3 H-dinitrophenyl group into enzyme cysteine residues was 0.96 mol per enzyme FAD for 68% conversion to the oxidase form. The modified enzyme was reconverted to the dehydrogenase form by incubation with dithiothreitol with concomitant release of 3 H-dinitrophenyl compounds. After modification with 3 H-FDNB followed by carboxymethylation under denaturating conditions, the enzyme was digested with proteases. Three 3 H-dinitrophenyl-labeled peptides were isolated and sequenced. The modified residues were identified to be Cys 535 , Cys 992 and Cys 1324 . These residues are conserved among the all known mammalian enzymes, but Cys 992 and Cys 1324 are not conserved in the chicken enzyme. Cys 1324 of the rat enzyme was found not to be involved in the conversion from the dehydrogenase to the oxidase by limited proteolysis experiments, but Cys 535 and Cys 992 which seemed to be modified alternatively with FDNB appear to be involved in the conversion. Xanthine oxidase (XO)1 catalyzes the oxidation of xanthine using molecular oxygen as an electron acceptor. The enzyme is a dimer containing one FAD, two 2Fe/2S centers, and one molybdopterin cofactor per M r 150,000 subunit (1, 2). Mammalian XO exists as the NAD-dependent dehydrogenase type (XDH) in freshly prepared samples, i.e. it exhibits low xanthine-O 2 reductase activity but high xanthine-NAD reductase activity even in the presence of O 2 (3, 4). But during extraction or purification procedures, the enzyme can be easily converted to an O 2 -dependent oxidase type (XO), i.e. it exhibits low xanthine-NAD activity but high xanthine-O 2 reductase activity. This conversion occurs reversibly by oxidation of sulfhydryl groups or irreversibly through proteolysis (3-10). As H 2 O 2 and O 2 Ϫ are formed as products when molecular oxygen is used as an electron acceptor, the conversion from XDH to XO has been proposed as the basis of the mechanism of recirculation injury (11).Although the enzyme is easily converted to XO, the enzyme can be purified as the dehydrogenase (XDH) form by rapid purification (6) or in the presence of thiol reagents such as mercaptoethanol or DTT (7, 8), or as an XO form which can be converted to XDH by incubation with thiols (9). The differences in structural, spectroscopic and kin...

cDNA Cloning, Sequencing, and Characterization of Male and Female Rat Liver Aldehyde Oxidase (rAOX1)

“…AOX does not have an NAD ϩ -dependent form of the enzyme, and therefore conversion between D-form and O-form is irrelevant. While cysteine residues critical for D-form to O-form conversion in XDH were not conserved in AOX (42), most of the 41 cysteine residues found in rat liver AOX are conserved with vertebrate XDHs. These cysteine residues would be expected to take part in the same biochemical reactivities as those found in XDH.…”

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

“…The mammalian XOR is synthesised as an NAD + -dependent dehydrogenase (throughout the text referred to as xanthine dehydrogenase, XD) but, although it transfers the electrons preferentially to NAD + , it can also catalyse electron transfer to O 2 . XD can, however, be readily converted to a ''strict'' oxidase form (named XO), either reversibly, through oxidation of the cysteine residues 535 and 992, or irreversibly, by proteolysis [5,6]. The cysteine oxidation (or proteolysis) causes a conformational change in the vicinity of the FAD, the site at which O 2 and NAD + react, increasing the midpoint potential of the flavin moiety and blocking the access of NAD + to FAD, but without disturbing the interactions between O 2 and FAD [7].…”

Unique amino acids cluster for switching from the dehydrogenase to oxidase form of xanthine oxidoreductase

“…Supporting the latter possibility are reports that four cysteine residues are modified when bovine XDH is converted to XO by incubation with dithiodipyridine (17). In addition, Cys-992 and Cys-535 were labeled in the rapid phase of the 1-fluoro-2,4-dinitrobenzene modification reaction, whereas another cysteine couple might be involved in the slower phase of the reaction that was observed (19).…”

“…Chemical modification studies have implicated Cys-535 and Cys-992 of rat liver (19) or bovine XOR (20) in the formation of a disulfide bond during the reversible XDH͞XO conversion. This interpretation has been challenged recently in a report describing gel chromatographic analyses of bovine enzyme in the XDH and proteolytically created XO forms (36).…”

“…Chemical modification studies have shown that Cys-535 and Cys-992 of rat liver XDH (19) or bovine XDH (20) are involved in the reversible conversion caused by modification of sulfhydryl residues by 1-fluoro-2,4-dinitrobenzene (19) or iodoacetamide (20). The crystal structure of XDH revealed that the FAD and Mo domains are connected by a linker consisting of amino acids 532-589, and this linker lies on the surface of the protein, spanning a distance of Ϸ70 Å.…”