Nitric oxide oxidises a ferrous mammalian lipoxygenase to a pre‐activated ferric species

Wiesner, Rainer; Rathmann, Jörg; Holzhütter, Hermann‐Georg; Stöβer, Reinhard; Mäder, Karsten; Nolting, H.‐F.; Kühn, Hartmut

doi:10.1016/0014-5793(96)00591-1

Cited by 36 publications

(26 citation statements)

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“…The kinetic data obtained during short-term incubation of the lipoxygenase with different concentrations of NO (Fig. 1) and EPR measurements [22] suggested the formation of an enzyme-NO complex. Since the kinetic lag phases of the progress curves in the presence of NO (Fig.…”

Section: Resultsmentioning

confidence: 78%

“…From Fig. 3 (trace A) it can be seen that the EPR spectrum of the native rabbit 15-lipoxygenase contains a weak and rather broad signal at g' = 4.3 suggesting that a small share of the enzyme was present in its ferric form [22]. After anaerobic short-term incubation of the enzyme with NO a strong signal at g' = 4.0 was observed, suggesting the formation of a ferrous lipoxygenase-NO complex.…”

Section: Resultsmentioning

confidence: 95%

“…Direct evidence for the formation of such an activated enzyme species was provided by EPR studies and X-ray absorption near-edge spectroscopy [22]. After long-term incubation, (>0.5 h) of the native enzyme with NO, three strong EPR signals at g' = 6.03,4.30 and 3.03 were detected all of which representing ferric iron species.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, these data suggest that long-term presence of NO causes the enzyme to be slowly converted to a transitionstate enzyme species which can be activated to the active ferric enzyme by hydroperoxy fatty acids. The assumption of such a transition-state enzyme is supported by EPR and XANES data indicating that NO is capable of oxidising the ferrous lipoxygenase to a ferric form [22]. These ferric lipoxygenase species may constitute the transition state [E'].…”

Section: Resultsmentioning

confidence: 99%

“…In fact, for the soybean lipoxygenase an inhibition of the enzyme by nitric oxide has been reported [7,201. This inhibition was accompanied by the formation of a ferrous lipoxygenase-NO complex which has been detected by EPR studies [19-211. Recently we have demonstrated that the mammalian 15-lipoxygenases from rabbit reticulocytes are a150 reversibly inhibited when incubated with NO [22]. Supi-isingly, the inhibition became less pronounced with increasing duration of the incubation period.…”

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A Kinetic Model for the Interaction of Nitric Oxide with a Mammalian Lipoxygenase

Holzhütter

Wiesner

Rathmann

et al. 1997

European Journal of Biochemistry

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Nitric oxide (NO) has been known for a long while to act as an inactivator of the soybean lipoxygenase-1 and cyclooxygenase. More recently, NO was shown to interact also with a mammalian 15-lipoxygenase [Wiesner, R., Rathmann, J., Holzhiitter, H. G., StoRer, R., Mader, K., Nolting, H. & Kiihn, H. (1996) Nitric oxide oxidises ferrous mammalian lipoxygenases to a pre-activated ferric species, FEBS Lett. 389, 229-2321. In this paper we present a detailed kinetic analysis of the interaction of NO with the 15-lipoxygenase from reticulocytes. Time courses of hydroperoxide formation were monitored after an anaerobic incubation of the enzyme with varying concentrations of NO and for varying length of the incubation period. Owing to the presence of O2 during the enzymatic reaction, the inhibitory effect of NO declines in a time-dependent manner since NO is converted into non-reactive NO, This is manifested as a lag phase of the time course. The lag phase becomes more pronounced if the enzyme is incubated over a fixed period (15 s) with increasing concentrations of NO. In contrast, increasing the length of the incubation period at fixed concentrations of NO diminishes the duration of the lag phase. These experimental findings can be accounted for by a kinetic model which assumes (a) fast reversible binding of NO to the inactive ferrous Fe(I1) form of the enzyme and (b) slow irreversible conversion of the Fe(I1)-NO complex into an activated ferric form of the enzyme which is susceptible to peroxide activation. The rate constants for these two different kinetic modes of NO action were estimated by fitting the solutions of the model equations to the experimental time courses. The dissociation constant of the Fe(I1)-NO complex amounts to 2.5 pM. Computer simulations performed on the basis of the model indicate that the response of the lipoxygenase to increasing intracellular NO concentrations depend upon the available peroxide tone: at low peroxide concentration the enzyme is initially trapped in the inactive ferrous form and thus may be activated by NO via the activated ferric species. On the other hand, at high peroxide concentrations, a partial inhibition of the initially active enzyme is expected.Keywords: lipoxygenase; nitric oxide ; kinetics ; mathematical modeling.Nitric oxide (NO) is a fairly stable radical exhibiting a variety of physiogical activities such as smooth muscle relaxation, inhibition of cell proliferation, inhibition of platelet aggregation and down regulation of the expression of adhesion molecules [I-51. In mammalian cells it is produced by an inducible and by a constitutively expressed nitric oxide synthase. The half-life of NO in aerobic solutions amounts to less than 10 s [6]. In vitro studies on the interaction of nitric oxide with metalloproteins indicated that it is capable of reacting with ferrous heme-and non-heme iron-containing proteins [7, 81 and thus may interfere with metabolic redox reactions. Lipoxygenases are non-heme iron-containing enzymes which catalyse the dioxygenation of polyen...

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Section: Resultsmentioning

confidence: 78%

Section: Resultsmentioning

confidence: 95%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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confidence: 93%

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