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Abstract Understanding the direct electron transfer processes between redox proteins and electrode surface is fundamental to understand the proteins mechanistic properties and for development of novel biosensors. In this study, nitric oxide reductase (NOR) extracted from Marinobacter hydrocarbonoclasticus bacteria was adsorbed onto a pyrolytic graphite electrode (PGE) to develop an unmediated enzymatic biosensor (PGE/NOR)) for characterization of NOR direct electrochemical behaviour and NOR electroanalytical features towards NO and O 2. Square-wave voltammetry showed the reduction potential of all the four NOR redox centers: 0.095 ± 0.002, −0.108 ± 0.008, −0.328 ± 0.001 and −0.635 ± 0.004 V vs. SCE for heme c , heme b , heme b 3 and non-heme Fe B , respectively. The determined sensitivity (−4.00 × 10−8 ± 1.84 × 10−9 A/μM and - 2.71 × 10−8 ± 1.44 × 10−9 A/μM for NO and O 2 , respectively), limit of detection (0.5 μM for NO and 1.0 μM for O 2) and the Michaelis Menten constant (2.1 and 7.0 μM for NO and O 2 , respectively) corroborated the higher affinity of NOR for its natural substrate (NO). No significant interference on sensitivity towards NO was perceived in the presence of O 2 , while the O 2 reduction was markedly and negatively impacted (3.6 times lower sensitivity) by the presence of NO. These results clearly demonstrate the high potential of NOR for the design of innovative NO biosensors. Highlights • Direct electron transfer of nitric oxide reductase (NOR) was described. • NOR bioelectrocatalytic activity towards NO and O 2 reduction was characterized. • Lower Michaelis Menten constant was obtained for NO than for O 2. • O 2 has no significant effect on NOR sensitivity towards NO reduction. • The unmediated NOR-based biosensor can be useful for NO sensing in biological events. [ABSTRACT FROM AUTHOR]

• Direct electrochemistry showed two signals assigned to CuA and CuZ(4Cu2S) centres. • CuA reduction potential is pH dependent in the presence of CuZ*(4Cu1S) • CuA centre is controlled by two proton-coupled electron transfer process. • "CuZ" reduction is controlled by a proton-coupled electron transfer process. • Enhanced catalytic potential consistent with the intermediate CuZ0(4Cu1S) Reduction of N 2 O to N 2 is catalysed by nitrous oxide reductase in the last step of the denitrification pathway. This multicopper enzyme has an electron transferring centre, CuA, and a tetranuclear copper-sulfide catalytic centre, "CuZ", which exists as CuZ*(4Cu1S) or CuZ(4Cu2S). The redox behaviour of these metal centres in Marinobacter hydrocarbonoclasticus nitrous oxide reductase was investigated by potentiometry and for the first time by direct electrochemistry. The reduction potential of CuA and CuZ(4Cu2S) was estimated by potentiometry to be +275 ± 5 mV and +65 ± 5 mV vs SHE, respectively, at pH 7.6. A proton-coupled electron transfer mechanism governs CuZ(4Cu2S) reduction potential, due to the protonation/deprotonation of Lys397 with a p K ox of 6.0 ± 0.1 and a p K red of 9.2 ± 0.1. The reduction potential of CuA, in enzyme samples with CuZ*(4Cu1S), is controlled by protonation of the coordinating histidine residues in a two-proton coupled electron transfer process. In the cyclic voltammograms, two redox pairs were identified corresponding to CuA and CuZ(4Cu2S), with no additional signals being detected that could be attributed to CuZ*(4Cu1S). However, an enhanced cathodic signal for the activated enzyme was observed under turnover conditions, which is explained by the binding of nitrous oxide to CuZ0(4Cu1S), an intermediate species in the catalytic cycle. [ABSTRACT FROM AUTHOR]