PH-DEPENDENCE FOR BINDING A SINGLE NITRITE ION TO EACH TYPE-2 COPPER CENTER IN THE COPPER-CONTAINING NITRITE REDUCTASE OF ALCALIGENES XYLOSOXIDANS

The first quantitative characterization of the interaction of NO2- wit h the Cu-containing dissimilatory nitrite reductase (NiR) of Alcaligen es xylosoxidans using steady-state kinetics, equilibrium gel filtratio n and EPR spectroscopy is described. Each molecule of this protein con sists of three equivalent subunits, each containing a type-1 Cu atom a nd also a type-2 Cu atom at each subunit interface. Enzyme activity in creased 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 filtratio n showed that binding of NO2- to the oxidized NiR was also pH-dependen t. 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 trim eric NiR molecule. Unexpectedly, NiR deficient in type-2 Cu centres bo und 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 c ontent. The NO2--induced changes in the EPR parameters of the type-2 C u 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 m u M (mean +/- S.E.M.) at pH 7.5 and < 30 mu M at pH 5.2. The apparent K-m for NO2- with saturating concentrations of dithionite as reductant was 35 mu M at pH 7.5, which is 10-fold tighter than for the oxidized enzyme, and is compatible with an ordered mechanism in which the enzy me is reduced before NO2- binds.