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Suicide inactivation of xanthine oxidoreductase during reduction of inorganic nitrite to nitric oxide.

ABSTRACT: Xanthine oxidoreductase (XOR) is progressively inactivated while catalysing the reduction of inorganic nitrite to NO by xanthine. Inactivation results from conversion of the enzyme into its desulpho-form. The rate of inactivation increases with nitrite concentration. Similar behaviour was shown when NADH replaced xanthine as reducing substrate. A kinetic model is proposed incorporating a 'suicide' inactivation involving an enzyme-substrate (product) complex, rather than inactivation by free NO. The model provides a good fit to progress curves of the reaction of xanthine or NADH with nitrite in the presence of the oxidase or dehydrogenase forms of the enzyme. Inorganic nitrate, like nitrite, was shown to be reduced at the molybdenum site of XOR. With xanthine as reducing substrate, nitrite was produced in essentially a 1:1 stoichiometric ratio with respect to urate. Unlike the case of nitrite, the enzyme was not significantly inactivated, implying that inactivation during nitrite reduction depends on the presence of nascent NO in its enzyme complex.

SUBMITTER: Godber BL

PROVIDER: S-EPMC1222064 | biostudies-other | 2001 Sep

REPOSITORIES: biostudies-other

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Project description:Nitrate reductases (NRs) are molybdoenzymes that reduce nitrate (NO3 -) to nitrite (NO2 -) in both mammals and plants. In mammals, the salival microbes take part in the generation of the NO2 - from NO3 -, which further produces nitric oxide (NO) either in acid-induced NO2 - reduction or in the presence of nitrite reductases (NiRs). Here, we report a new approach of VCl3 (V3+ ion source) induced step-wise reduction of NO3 - in a CoII-nitrato complex, [(12-TMC)CoII(NO3 -)]+ (2,{CoII-NO3 -}), to a CoIII-nitrosyl complex, [(12-TMC)CoIII(NO)]2+ (4,{CoNO}8), bearing an N-tetramethylated cyclam (TMC) ligand. The VCl3 inspired reduction of NO3 - to NO is believed to occur in two consecutive oxygen atom transfer (OAT) reactions, i.e., OAT-1 = NO3 - → NO2 - (r1) and OAT-2 = NO2 - → NO (r2). In these OAT reactions, VCl3 functions as an O-atom abstracting species, and the reaction of 2 with VCl3 produces a CoIII-nitrosyl ({CoNO}8) with VV-Oxo ({VV[double bond, length as m-dash]O}3+) species, via a proposed CoII-nitrito (3, {CoII-NO2 -}) intermediate species. Further, in a separate experiment, we explored the reaction of isolated complex 3 with VCl3, which showed the generation of 4 with VV-Oxo, validating our proposed reaction sequences of OAT reactions. We ensured and characterized 3 using VCl3 as a limiting reagent, as the second-order rate constant of OAT-2 (k 2 /) is found to be ∼1420 times faster than that of the OAT-1 (k 2) reaction. Binding constant (K b) calculations also support our proposition of NO3 - to NO transformation in two successive OAT reactions, as K b(CoII-NO2 -) is higher than K b(CoII-NO3 -), hence the reaction moves in the forward direction (OAT-1). However, K b(CoII-NO2 -) is comparable to K b{CoNO}8 , and therefore sequenced the second OAT reaction (OAT-2). Mechanistic investigations of these reactions using 15N-labeled-15NO3 - and 15NO2 - revealed that the N-atom in the {CoNO}8 is derived from NO3 - ligand. This work highlights the first-ever report of VCl3 induced step-wise NO3 - reduction (NRs activity) followed by the OAT induced NO2 - reduction and then the generation of Co-nitrosyl species {CoNO}8.

Project description:Cardiovascular disease is the leading cause of death and disability worldwide with increased oxidative stress and reduced NO bioavailability serving as key risk factors. For decades, elevation in protein abundance and enzymatic activity of xanthine oxidoreductase (XOR) under hypoxic/inflammatory conditions has been associated with organ damage and vascular dysfunction. Recent reports have challenged this dogma by identifying a beneficial function for XOR, under similar hypoxic/acidic conditions, whereby XOR catalyzes the reduction of nitrite (NO2-) to nitric oxide (NO) through poorly defined mechanisms. We previously reported that hydrogen sulfide (H2S/sulfide) confers significant vascular benefit under these same conditions via NO2- mediated mechanisms independent of nitric oxide synthase (NOS). Here we report for the first time the convergence of H2S, XOR, and nitrite to form a concerted triad for NO generation. Specifically, hypoxic endothelial cells show a dose-dependent, sulfide and polysulfide (diallyl trisulfide (DATS)-induced, NOS-independent NO2- reduction to NO that is dependent upon the enzymatic activity of XOR. Interestingly, nitrite reduction to NO was found to be slower and more sustained with DATS compared to H2S. Capacity for sulfide/polysulfide to produce an XOR-dependent impact on NO generation translates to salutary actions in vivo as DATS administration in cystathionine-γ-lyase (CSE) knockout mice significantly improved hindlimb ischemia blood flow post ligation, while the XOR-specific inhibitor, febuxostat (Febx), abrogated this benefit. Moreover, flow-mediated vasodilation (FMD) in CSE knockout mice following administration of DATS resulted in greater than 4-fold enhancement in femoral artery dilation while co-treatment with Febx completely completely abrogated this effect. Together, these results identify XOR as a focal point of convergence between sulfide- and nitrite-mediated signaling, as well as affirm the critical need to reexamine current dogma regarding inhibition of XOR in the context of vascular dysfunction.

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Suicide inactivation of xanthine oxidoreductase during reduction of inorganic nitrite to nitric oxide.

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