Proton-coupled electron transfer mechanisms of the copper centres of nitrous oxide reductase from Marinobacter hydrocarbonoclasticus – An electrochemical study

Abstract

Reduction of N2O to N2 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 pKox of 6.0±0.1 and a pKred 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 CuZ 0 (4Cu1S), an intermediate species in the catalytic cycle.

Disclosing the redox metabolism of drugs: The essential role of electrochemistry

“…Accordingly, iron-centered hydrogenases have been reconstructed at electrodes and their reactivity explored electrochemically [24,25]. Alternatively, mimicry strategies are developed featuring more simple species possessing the reactive core metal-ligand structure, to address general reactivity points, namely fundamental aspects of proton coupled electron transfers [26][27][28]. Recent publications also focus on the general reactivity of cytochrome C, a key-electron relaying hemoprotein involved in homeostasis and apoptosis processes [29,30].…”

The effect of pH on Marinobacter hydrocarbonoclasticus denitrification pathway and nitrous oxide reductase

“…CuZ(4Cu2S) can be isolated in two oxidation states, [2Cu 2+ -2Cu 1+ ] or [1Cu 2+ -3Cu 1+ ], with the reduction potential of this redox pair being + 60 mV, at pH 7.5 [18,25], but cannot be reduced to the [4Cu 1+ ] oxidation state [16,25]. In its reduced state, CuZ(4Cu2S) can react with N2O but has a very low turnover number (kcat = 0.6 h -1 ) [16].…”

Histidine-Gated Proton-Coupled Electron Transfer to the CuA Site of Nitrous Oxide Reductase

“…The derived p K a value of 6.19 in the lower pH range was assigned to the protonation of a histidine side chain . A most recent report by Pauleta and Moura showed a near-perfect Nernstian pH-dependence of the reduction potential ( E 0 ) of the Cu A site in Mh N 2 OR. , Taking into account the interference from Cu Z , this was interpreted as a 2H + /e – process for Cu A in Mh N 2 OR . A cyclic voltammetry analysis of the Cu A site of engineered azurin showed a decrease of approximately 180 mV in reduction potential upon a shift from pH 4 to pH 7, and the authors assigned H120 (Figure S1) as the site of protonation that accompanies electron transfer, although H46 (the equivalent to H583 in Ps N 2 OR) was not completely ruled out .…”

“…62,63 Taking into account the interference from Cu Z , this was interpreted as a 2H + /e − process for Cu A in MhN 2 OR. 64 A cyclic voltammetry analysis of the Cu A site of engineered azurin showed a decrease of approximately 180 mV in reduction potential upon a shift from pH 4 to pH 7, and the authors assigned H120 (Figure S1) as the site of protonation that accompanies electron transfer, although H46 (the equivalent to H583 in PsN 2 OR) was not completely ruled out. 65 However, the Cu A site of TtCOX did not show any dependence of its reduction potential on pH in the physiologically relevant range.…”