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.
Protonation state of the Cu4S2CuZsite in nitrous oxide reductase: redox dependence and insight into reactivity