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. 1997 Jun 1;324(Pt 2):511–516. doi: 10.1042/bj3240511

pH-dependence for binding a single nitrite ion to each type-2 copper centre in the copper-containing nitrite reductase of Alcaligenes xylosoxidans.

Z H Abraham 1, B E Smith 1, B D Howes 1, D J Lowe 1, R R Eady 1
PMCID: PMC1218459  PMID: 9182711

Abstract

The first quantitative characterization of the interaction of NO2(-) with the Cu-containing dissimilatory nitrite reductase (NiR) of Alcaligenes xylosoxidans using steady-state kinetics, equilibrium gel filtration and EPR spectroscopy is described. Each molecule of this protein consists of three equivalent subunits, each containing a type-1 Cu atom and also a type-2 Cu atom at each subunit interface. Enzyme activity increased in a biphasic manner with decreasing pH, having an optimum at pH 5.2 and a plateau between pH 6.1 and 5.8. Equilibrium gel filtration showed that binding of NO2(-) to the oxidized NiR was also pH-dependent. At pH 7.5, no binding was detectable, but binding was detectable at lower pH values. At pH 5.2, the concentration-dependence for binding of NO2(-) to the enzyme showed that approx. 4.1 NO2(-) ions bound per trimeric NiR molecule. Unexpectedly, NiR deficient in type-2 Cu centres bound 1.3 NO2(-) ions per trimer. When corrected for this binding, a value of 3 NO2(-) ions bound per trimer of NiR, equivalent to the type-2 Cu content. The NO2(-)-induced changes in the EPR parameters of the type-2 Cu centre of the oxidized enzyme showed a similar pH-dependence to that of the activity. Binding constants for NO2(-) at a single type of site, after allowing for the non-specifically bound NO2(-), were 350+/-35 microM (mean+/-S.E.M.) at pH 7.5 and <30 microM at pH 5.2. The apparent Km for NO2(-) with saturating concentrations of dithionite as reductant was 35 microM at pH 7.5, which is 10-fold tighter than for the oxidized enzyme, and is compatible with an ordered mechanism in which the enzyme is reduced before NO2(-) binds.

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Selected References

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