Biochemical characterization of the purple form of Marinobacter hydrocarbonoclasticus nitrous oxide reductase

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.