Abstract
Nitrous oxide reductase (N(2)OR) catalyses the final step of the denitrification pathway-the reduction of nitrous oxide to nitrogen. The catalytic centre (CuZ) is a unique tetranuclear copper centre bridged by inorganic sulphur in a tetrahedron arrangement that can have different oxidation states. Previously, Marinobacter hydrocarbonoclasticus N(2)OR was isolated with the CuZ centre as CuZ*, in the [1Cu(2+) : 3Cu(+)] redox state, which is redox inert and requires prolonged incubation under reductive conditions to be activated. In this work, we report, for the first time, the isolation of N(2)OR from M. hydrocarbonoclasticus in the 'purple' form, in which the CuZ centre is in the oxidized [2Cu(2+) : 2Cu(+)] redox state and is redox active. This form of the enzyme was isolated in the presence of oxygen from a microaerobic culture in the presence of nitrate and also from a strictly anaerobic culture. The purple form of the enzyme was biochemically characterized and was shown to be a redox active species, although it is still catalytically non-competent, as its specific activity is lower than that of the activated fully reduced enzyme and comparable with that of the enzyme with the CuZ centre in either the [1Cu(2+) : 3Cu(+)] redox state or in the redox inactive CuZ* state.