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

• Published in: Biochemical journal Vol. 324; no. 2; pp. 511 - 516
• Main Authors: Abraham, Z.H.L, Smith, B.E, Howes, B.D, Lowe, D.J, Eady, R.R
• Format: Journal Article
• Language: English
• Published: England 01.06.1997
• Subjects: Alcaligenes - enzymology; Alcaligenes xylosoxidans; Bacterial Proteins - metabolism; Copper - metabolism; Electron Spin Resonance Spectroscopy; Hydrogen-Ion Concentration; Nitrite Reductases - metabolism; Oxidation-Reduction; plant biochemistry; plant physiology; Protein Binding
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3240511

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 micromolar (mean +/- S.E.M.) at pH 7.5 and < 30 micromolar at pH 5.2. The apparent Km for NO2(-) with saturating concentrations of dithionite as reductant was 35 micromolar 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.